Gerhard Schenk

High resolution crystal structure of a fluoride-inhibited organophosphate-degrading metallohydrolase

Publisher: Elsevier BV

Date: 12-2017

DOI: 10.1016/J.JINORGBIO.2017.06.013

Abstract: Metal ion-dependent, organophosphate-degrading enzymes (OP hydrolases) have received increasing attention due to their ability to degrade and thus detoxify commonly used pesticides and nerve agents such as sarin and VX. These enzymes thus garner strong potential as bioremediators. The OP hydrolase from Agrobacterium radiobacter (OpdA) is one of the most efficient members of this group of enzymes. Previous studies have indicated that the choice of the hydrolysis-initiating nucleophile may depend on the pH of the reaction, with a metal ion-bridging hydroxide being preferred at lower pH (i.e. pH≤8.5), and a terminally coordinated hydroxide at higher pH (i.e. pH>9.0). Furthermore, fluoride was shown to be a potent inhibitor of the reaction, but only at low pH. Here, the crystal structure (1.3Å, pH6) of OpdA in presence of fluoride is described. While the first coordination sphere in the active site displays minimal changes in the presence of fluoride, the hydrogen bonding network that connects the dimetallic metal center to the substrate binding pocket is disrupted. Thus, the structure of fluoride-inhibited OpdA demonstrates the significance of this hydrogen bond network in controlling the mechanism and function of this enzyme.

Discovery of a Pyrimidinedione Derivative with Potent Inhibitory Activity against Mycobacterium tuberculosis Ketol–Acid Reductoisomerase

Publisher: Wiley

Date: 12-01-2020

DOI: 10.1002/CHEM.202004665

Structural elements that modulate the substrate specificity of plant purple acid phosphatases: avenues for improved phosphorus acquisition in crops

Publisher: Cold Spring Harbor Laboratory

Date: 28-08-2019

DOI: 10.1101/749564

Abstract: Phosphate acquisition by plants is an essential process that is directly implicated in the optimization of crop yields. Purple acid phosphatases (PAPs) are ubiquitous metalloenzymes, which catalyze the hydrolysis of a wide range of phosphate esters and anhydrides. While some plant PAPs display a preference for ATP as the substrate, others are efficient in hydrolyzing phytate or 2-phosphoenolpyruvate (PEP). PAP from red kidney bean (rkbPAP) is an efficient ATP- and ADPase, but has no activity towards phytate. The crystal structure of this enzyme in complex with an ATP analogue (to 2.20 Å resolution) provides insight into the amino acid residues that play an essential role in binding this substrate. Homology modelling was used to generate three-dimensional structures for the active sites of PAPs from tobacco (NtPAP) and Arabidopsis thaliana (AtPAP12 and AtPAP26) that are efficient in hydrolyzing phytate and PEP as substrates, respectively. In combination with substrate docking simulations and a phylogenetic analysis of 49 plant PAP sequences (including the first PAP sequences reported from Eucalyptus ), several active site residues were identified that are important in defining the substrate specificities of plant PAPs. These results may inform bioengineering studies aimed at identifying and incorporating suitable plant PAP genes into crops to improve phosphorus use efficiency. Organic phosphorus sources increasingly supplement or replace inorganic fertilizer, and efficient phosphorus use of crops will lower the environmental footprint of agriculture while enhancing food production.

Synthesis, characterization and computational investigation of the phosphatase activity of a dinuclear Zinc(II) complex containing a new heptadentate asymmetric ligand

Publisher: Elsevier BV

Date: 02-2023

DOI: 10.1016/J.JINORGBIO.2022.112064

Abstract: We report the synthesis of a new asymmetric heptadentate ligand based on the 1,3-diaminopropan-2-ol backbone. The ligand 3-[[3-(bis-pyridin-2-ylmethyl-amino)-2-hydroxy-propyl]-(2-carbamoyl-ethyl)-amino]-propionamide (HL1) contains two amide and two pyridine groups attached to the 1,3-diaminopropan-2-ol core. Reaction between HL1 and Zn(ClO

Identification and characterization of an unusual metallo-β-lactamase from Serratia proteamaculans

Publisher: Springer Science and Business Media LLC

Date: 28-08-2013

DOI: 10.1007/S00775-013-1035-Z

Abstract: Metallo-β-lactamases (MBLs) are a family of metalloenzymes that are capable of hydrolyzing β-lactam antibiotics and are an important means by which bacterial pathogens use to inactivate antibiotics. A database search of the available amino acid sequences from Serratia proteamaculans indicates the presence of an unusual MBL. A full length amino acid sequence alignment indicates overall homology to B3-type MBLs, but also suggests considerable variations in the active site, notably among residues that are relevant to metal ion binding. Steady-state kinetic measurements further indicate functional differences and identify two relevant pK a values for catalysis (3.8 for the enzyme-substrate complex and 7.8 for the free enzyme) and a preference for penams with modest reactivity towards some cephalosporins. An analysis of the metal ion content indicates the presence of only one zinc ion per active site in the resting enzyme. In contrast, kinetic data suggest that the enzyme may operate as a binuclear enzyme, and it is thus proposed that a catalytically active di-Zn(2+) center is formed only once the substrate is present.

Metal-Ion Mutagenesis: Conversion of a Purple Acid Phosphatase from Sweet Potato to a Neutral Phosphatase with the Formation of an Unprecedented Catalytically Competent Mn^IIMn^II

Publisher: American Chemical Society (ACS)

Date: 26-05-2009

DOI: 10.1021/JA900797U

Abstract: The currently accepted paradigm is that the purple acid phosphatases (PAPs) require a heterovalent, dinuclear metal-ion center for catalysis. It is believed that this is an essential feature for these enzymes in order for them to operate under acidic conditions. A PAP from sweet potato is unusual in that it appears to have a specific requirement for manganese, forming a unique Fe(III)-mu-(O)-Mn(II) center under catalytically optimal conditions (Schenk et al. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 273). Herein, we demonstrate, with detailed electron paramagnetic resonance (EPR) spectroscopic and kinetic studies, that in this enzyme the chromophoric Fe(III) can be replaced by Mn(II), forming a catalytically active, unprecedented antiferromagnetically coupled homo alent Mn(II)-mu-(H)OH-mu-carboxylato-Mn(II) center in a PAP. However, although the enzyme is still active, it no longer functions as an acid phosphatase, having optimal activity at neutral pH. Thus, PAPs may have evolved from distantly related alent dinuclear metallohydrolases that operate under pH neutral conditions by stabilization of a trivalent- alent metal-ion core. The present Mn(II)-Mn(II) system models these distant relatives, and the results herein make a significant contribution to our understanding of the role of the chromophoric metal ion as an activator of the nucleophile. In addition, the detailed analysis of strain broadened EPR spectra from exchange-coupled dinuclear Mn(II)-Mn(II) centers described herein provides the basis for the full interpretation of the EPR spectra from other dinuclear Mn metalloenzymes.

Realising the Circular Phosphorus Economy delivers for Sustainable Development Goals

Publisher: Wiley

Date: 18-03-2022

DOI: 10.1002/ESSOAR.10510868.1

Second-Sphere Effects in Dinuclear Fe^IIIZn^II Hydrolase Biomimetics: Tuning Binding and Reactivity Properties

Publisher: American Chemical Society (ACS)

Date: 13-12-2017

DOI: 10.1021/ACS.INORGCHEM.7B02384

Abstract: Herein, we report the synthesis and characterization of two dinuclear Fe

Effect of Chemically Distinct Substrates on the Mechanism and Reactivity of a Highly Promiscuous Metallohydrolase

Publisher: American Chemical Society (ACS)

Date: 24-02-2020

DOI: 10.1021/ACSCATAL.9B04847

Crystal structure of Mycobacterium tuberculosis ketol‐acid reductoisomerase at 1.0 Å resolution – a potential target for anti‐tuberculosis drug discovery

Publisher: Wiley

Date: 18-02-2016

DOI: 10.1111/FEBS.13672

Abstract: The biosynthetic pathway for the branched-chain amino acids is present in plants, fungi and bacteria, but not in animals, making it an attractive target for herbicidal and antimicrobial drug discovery. Ketol-acid reductoisomerase (KARI EC 1.1.1.86) is the second enzyme in this pathway, converting in a Mg(2+) - and NADPH-dependent reaction either 2-acetolactate or 2-aceto-2-hydroxybutyrate to their corresponding 2,3-dihydroxy-3-alkylbutyrate products. Here, we have determined the crystal structure of Mycobacterium tuberculosis (Mt) KARI, a class I KARI, with two magnesium ions bound in the active site. X-ray data were obtained to 1.0 Å resolution and the final model has an Rfree of 0.163. The structure shows that the active site is solvent-accessible with the two metal ions separated by 4.7 Å. A comparison of this structure with that of Mg(2+) -free Pseudomonas aeruginosa KARI suggests that upon magnesium binding no movement of the N domain relative to the C domain occurs. However, upon formation of the Michaelis complex, as illustrated in the structure of Slackia exigua KARI in complex with NADH.Mg(2+) . N-hydroxy-N-isopropyloxamate (IpOHA, a transition state analog), domain movements and reduction of the metal-metal distance to 3.5 Å are observed. This inherent flexibility therefore appears to be critical for initiation of the KARI-catalyzed reaction. This study provides new insights into the complex structural rearrangements required for activity of KARIs, particularly those belonging to class I, and provides the framework for the rational design of Mt KARI inhibitors that can be tested as novel antituberculosis agents. Coordinates and structure factors for the Mt KARI.Mg(2+) complex are available in the Protein Data Bank under accession number 4YPO.

The applications of binuclear metallohydrolases in medicine: Recent advances in the design and development of novel drug leads for purple acid phosphatases, metallo-β-lactamases and arginases

Publisher: Elsevier BV

Date: 04-2014

DOI: 10.1016/J.EJMECH.2014.02.008

Abstract: Binuclear metallohydrolases are a family of proteins that can be targeted for drug discovery. The common feature of these enzymes is the presence of two closely spaced metal ions (i.e. less than 4 Å apart) that capture a water molecule that is used as a nucleophile in highly specific hydrolytic reactions. In this mini-review we describe what is known about the biological and catalytic activity, three-dimensional structure and inhibition for three prominent drug targets in this family of enzymes, (i) purple acid phosphatases, (ii) metallo-β-lactamases and (iii) arginases. These enzymes are targets for the development of chemotherapeutics to treat a range of disorders including osteoporosis, cardiovascular disease and erectile dysfunctions, but also to stem the spread of antibiotic resistance, a major threat to global health care.

Translational fusion of terpene synthases enhances metabolic flux by increasing protein stability

Publisher: Cold Spring Harbor Laboratory

Date: 09-11-2022

DOI: 10.1101/2022.11.08.515726

Abstract: The end-to-end fusion of enzymes that catalyse successive steps in a reaction pathway is a metabolic engineering strategy that has been successfully applied in a variety of pathways and is particularly common in terpene bioproduction. Despite its popularity, limited work has been done to interrogate the mechanism of metabolic enhancement from enzyme fusion. We observed a remarkable -fold improvement in nerolidol production upon translational fusion of nerolidol synthase (a sesquiterpene synthase) to farnesyl diphosphate synthase. This delivered a titre increase from 29.6 mg/L up to 4.2 g/L nerolidol in a single engineering step. Whole-cell proteomic analysis revealed that nerolidol synthase levels in the fusion strains were greatly elevated compared to the non-fusion control. Similarly, the fusion of nerolidol synthase to non-catalytic domains also produced comparable increases in titre, which coincided with improved enzyme expression. When farnesyl diphosphate synthase was fused to other terpene synthases, we observed more modest improvements in terpene titre (1.9- and 3.8-fold), which corresponds to increases of a similar magnitude in terpene synthase expression. Therefore, increased in vivo enzyme levels – resulting from improved expression and/or stability – is likely to be a major driver of catalytic enhancement from enzyme fusion.

Dolutegravir plus lamivudine versus dolutegravir plus tenofovir disoproxil fumarate and emtricitabine in antiretroviral-naive adults with HIV-1 infection (GEMINI-1 and GEMINI-2): week 48 results from

Publisher: Elsevier BV

Date: 2019

DOI: 10.1016/S0140-6736(18)32462-0

An Unprecedented Fe^III(μ-OH)Zn^II Complex that Mimics the Structural and Functional Properties of Purple Acid Phosphatases

Publisher: American Chemical Society (ACS)

Date: 23-05-2007

DOI: 10.1021/JA071184L

A structural and catalytic model for zinc phosphoesterases

Publisher: Royal Society of Chemistry (RSC)

Date: 2008

DOI: 10.1039/B806391E

Abstract: A structural model for the active site of phosphoesterases, enzymes that degrade organophosphate neurotoxins, has been synthesised. The ligand [2-((2-hydroxy-3-(((2-hydroxyethyl)(pyridin-2-ylmethyl)amino)methyl)-5-methylbenzyl)(pyridin-2-ylmethyl)amino)acetic acid] (H(3)L1) and two Zn(ii) complexes have been prepared and characterised as [Zn(2)(HL1)(CH(3)COO)](PF(6)).H(2)O and Li[Zn(2)(HL1)](4)(PO(4))(2)(PF(6))(3).(CH(3)OH). The ligand (H(3)L1) and complex [Zn(2)(HL1)(CH(3)COO)](PF(6)).H(2)O were characterised through (1)H NMR, (13)C NMR, mass spectroscopy and microanalysis. The X-ray crystal structure of Li[Zn(2)(HL1)](4)(PO(4))(2)(PF(6))(3).(CH(3)OH) revealed a tetramer of dinuclear complexes, bridged by two phosphate molecules and bifurcating acetic acid arms. Functional studies of the zinc complex with the substrate bis(4-nitrophenyl)phosphate (bNPP) determined the complex with HL1(2-) to be a competent catalyst with k(cat) = 1.26 +/- 0.06 x 10(-6) s(-1).

Deacidification of grass silage press juice by continuous production of acetoin from its lactate via an immobilized enzymatic reaction cascade

Publisher: Elsevier BV

Date: 12-2017

DOI: 10.1016/J.BIORTECH.2017.08.203

Abstract: An immobilized enzymatic reaction cascade was designed and optimized for the deacidification of grass silage press juice (SPJ), thus facilitating the production of bio-based chemicals. The cascade involves a three-step process using four enzymes immobilized in a Ca-alginate gel and uses lactic acid to form acetoin, a value-added product. The reaction is performed with a continuous, pH-dependent substrate feed under oxygenation. With titrated lactic acid yields of up to 91% and reaction times of ca. 6h was achieved. Using SPJ as titrant yields of 49% were obtained within 6h. In this deacidification process, with acetoin one value-added bio-based chemical is produced while simultaneously the remaining press juice can be used in applications that require a higher pH. Such, this system can be applied in a multi-product biorefinery concept to take full advantage of nutrient-rich SPJ, which is a widely available and easily storable renewable resource.

The use of SWATH to analyse the dynamic changes of bacterial proteome of carbapanemase-producing Escherichia coli under antibiotic pressure

Publisher: Springer Science and Business Media LLC

Date: 03-2018

DOI: 10.1038/S41598-018-21984-9

Abstract: Antibiotic resistance associated with the clinically significant carbapenemases KPC, NDM and OXA-48 in Enterobacteriaceae is emerging as worldwide. In Australia, IMP-producing Enterobacteriaceae are the most prevalent carbapenemase-producing Enterobacteriaceae (CPE). Genomic characteristics of such CPE are well described, but the corresponding proteome is poorly characterised. We have thus developed a method to analyse dynamic changes in the proteome of CPE under antibiotic pressure. Specifically, we have investigated the effect of meropenem at sub-lethal concentrations to develop a better understanding of how antibiotic pressure leads to resistance. Escherichia coli strains producing either NDM-, IMP- or KPC-type carbapenemases were included in this study, and their proteomes were analysed in growth conditions with or without meropenem. The most significant difference in the bacterial proteomes upon the addition of meropenem was triggered amongst NDM-producers and to a lower extent amongst KPC-producers. In particular, HU DNA-binding proteins, the GroEL/GroES chaperonin complex and GrpE proteins were overexpressed. These proteins may thus contribute to the better adaptability of NDM- and KPC-producers to meropenem. A significant meropenem-induced increase in the expression of the outer membrane protein A was only observed in IMP-producers, thus demonstrating that carbapenemase-mediated resistance relies on far more complex mechanisms than simple inactivation of the antibiotic.

Biotechnological potential and initial characterization of two novel sesquiterpene synthases from Basidiomycota Coniophora puteana for heterologous production of δ-cadinol

Publisher: Springer Science and Business Media LLC

Date: 19-04-2022

DOI: 10.1186/S12934-022-01791-8

Abstract: Terpene synthases are versatile catalysts in all domains of life, catalyzing the formation of an enormous variety of different terpenoid secondary metabolites. Due to their erse bioactive properties, terpenoids are of great interest as innovative ingredients in pharmaceutical and cosmetic applications. Recent advances in genome sequencing have led to the discovery of numerous terpene synthases, in particular in Basidiomycota like the wood rotting fungus Coniophora puteana , which further enhances the scope for the manufacture of terpenes for industrial purposes. In this study we describe the identification of two novel (+)-δ-cadinol synthases from C. puteana, Copu5 and Copu9. The sesquiterpene (+)-δ-cadinol was previously shown to exhibit cytotoxic activity therefore having an application as possible, new, and sustainably sourced anti-tumor agent. In an Escherichia coli strain, optimized for sesquiterpene production, titers of 225 mg l −1 and 395 mg l −1 , respectively, could be achieved. Remarkably, both enzymes share the same product profile thereby representing the first two terpene synthases from Basidiomycota with identical product profiles. We solved the crystal structure of Copu9 in its closed conformation, for the first time providing molecular details of sesquiterpene synthase from Basidiomycota. Based on the Copu9 structure, we conducted structure-based mutagenesis of amino acid residues lining the active site, thereby altering the product profile. Interestingly, the mutagenesis study also revealed that despite the conserved product profiles of Copu5 and Copu9 different conformational changes may accompany the catalytic cycle of the two enzymes. This observation suggests that the involvement of tertiary structure elements in the reaction mechanism(s) employed by terpene synthases may be more complex than commonly expected. The presented product selectivity and titers of Copu5 and Copu9 may pave the way towards a sustainable, biotechnological production of the potentially new bioactive (+)-δ-cadinol. Furthermore, Copu5 and Copu9 may serve as model systems for further mechanistic studies of terpenoid catalysis.

Biomimetics for purple acid phosphatases: A historical perspective

Publisher: Elsevier BV

Date: 2023

DOI: 10.1016/J.JINORGBIO.2022.112061

Abstract: Biomimetics hold potential for varied applications in biotechnology and medicine but have also attracted particular interest as benchmarks for the functional study of their more complex biological counterparts, e.g. metalloenzymes. While many of the synthetic systems adequately mimic some structural and functional aspects of their biological counterparts the catalytic efficiencies displayed are mostly far inferior due to the smaller size and the associated lower complexity. Nonetheless they play an important role in bioinorganic chemistry. Numerous ex les of biologically inspired and informed artificial catalysts have been reported, designed to mimic a plethora of chemical transformations, and relevant ex les are highlighted in reviews and scientific reports. Herein, we discuss biomimetics of the metallohydrolase purple acid phosphatase (PAP), ex les of which have been used to showcase synergistic research advances for both the biological and synthetic systems. In particular, we focus on the seminal contribution of our colleague Prof. Ademir Neves, and his group, pioneers in the design and optimization of suitable ligands that mimic the active site of PAP.

Ligand modifications modulate the mechanism of binuclear phosphatase biomimetics

Publisher: Elsevier BV

Date: 03-2013

DOI: 10.1016/J.POLY.2012.06.008

Immobilization of the enzyme GpdQ on magnetite nanoparticles for organophosphate pesticide bioremediation

Publisher: Elsevier BV

Date: 02-2014

DOI: 10.1016/J.JINORGBIO.2013.10.007

Abstract: Annually thousands of people die or suffer from organophosphate (pesticide) poisoning. In order to remove these toxic compounds from the environment, the use of enzymes as bioremediators has been proposed. We report here a Ser127Ala mutant based on the enzyme glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes. The mutant, with improved metal binding abilities, has been immobilized using glutaraldehyde on PAMAM dendrimer-modified magnetite nanoparticles. The immobilized system was characterized using elemental analysis as well as infrared, transmission electron and X-ray photoelectron spectroscopies. The amount of GpdQ that was immobilized with the optimized procedure was 1.488 nmol per g MNP. A kinetic assay has been designed to evaluate the activity of the system towards organophosphoester substrates. The specific activity towards BPNPP directly after immobilization was 3.55 μmol mg(-1)min(-1), after one week 3.39 μmol mg(-1)min(-1) and after 120 days 3.36 μmol mg(-1)min(-1), demonstrating that the immobilized enzyme was active for multiple cycles and could be stored on the nanoparticles for a prolonged period.

Bacterial and Plant Ketol-Acid Reductoisomerases Have Different Mechanisms of Induced Fit during the Catalytic Cycle

Publisher: Elsevier BV

Date: 12-2012

DOI: 10.1016/J.JMB.2012.09.018

Abstract: Ketol-acid reductoisomerase (KARI) is the second enzyme in the branched-chain amino acid biosynthesis pathway, which is found in plants, fungi and bacteria but not in animals. This difference in metabolism between animals and microorganisms makes KARI an attractive target for the development of antimicrobial agents. Herein we report the crystal structure of Escherichia coli KARI in complex with Mg(2+) and NADPH at 2.3Å resolution. Ultracentrifugation studies confirm that the enzyme exists as a tetramer in solution, and isothermal titration calorimetry shows that the binding of Mg(2+) increases structural disorder while the binding of NADPH increases the structural rigidity of the enzyme. Comparison of the structure of the E. coli KARI-Mg(2+)-NADPH complex with that of enzyme in the absence of cofactors shows that the binding of Mg(2+) and NADPH opens the interface between the N- and C-domains, thereby allowing access for the substrates to bind: the existence of only a small opening between the domains in the crystal structure of the unliganded enzyme signifies restricted access to the active site. This observation contrasts with that in the plant enzyme, where the N-domain can rotate freely with respect to the C-domain until the binding of Mg(2+) and/or NADPH stabilizes the relative positions of these domains. Support is thereby provided for the idea that plant and bacterial KARIs have evolved different mechanisms of induced fit to prepare the active site for catalysis.

Detailed stratified GWAS analysis for severe COVID-19 in four European populations

Publisher: Cold Spring Harbor Laboratory

Date: 23-07-2021

DOI: 10.1101/2021.07.21.21260624

Abstract: Given the highly variable clinical phenotype of Coronavirus disease 2019 (COVID-19), a deeper analysis of the host genetic contribution to severe COVID-19 is important to improve our understanding of underlying disease mechanisms. Here, we describe an extended GWAS meta-analysis of a well-characterized cohort of 3,260 COVID-19 patients with respiratory failure and 12,483 population controls from Italy, Spain, Norway and Germany/Austria, including stratified analyses based on age, sex and disease severity, as well as targeted analyses of chromosome Y haplotypes, the human leukocyte antigen (HLA) region and the SARS-CoV-2 peptidome. By inversion imputation, we traced a reported association at 17q21.31 to a highly pleiotropic ∼0.9-Mb inversion polymorphism and characterized the potential effects of the inversion in detail. Our data, together with the 5 th release of summary statistics from the COVID-19 Host Genetics Initiative, also identified a new locus at 19q13.33, including NAPSA , a gene which is expressed primarily in alveolar cells responsible for gas exchange in the lung.

Dinuclear Cobalt(II) Complexes as Metallo‐β‐lactamase Mimics

Publisher: Wiley

Date: 30-04-2013

DOI: 10.1002/EJIC.201300280

Phosphate ester cleavage promoted by a tetrameric iron(III) complex

Publisher: Springer Science and Business Media LLC

Date: 28-08-2011

DOI: 10.1007/S00775-010-0696-0

Abstract: The purple acid phosphatases (PAPs) are the only binuclear metallohydrolases where the necessity for a heterovalent active site [Fe(III)-M(II) (M is Fe, Zn or Mn)] for catalysis has been established. The paradigm for the construction of PAP biomimetics, both structural and functional, is that the ligands possess characteristics which mimic those of the donor sites of the metalloenzyme and permit discrimination between trivalent and alent metal ions. The donor atom set of the ligand 2-((2-hydroxy-5-methyl-3-((pyridin-2-ylmethylamino)methyl)benzyl)(2-hydroxybenzyl)amino)acetic acid (H(3)HPBA) mimics that of the active site of PAP although the iron(III) complex of this ligand has been characterized as the tetramer [Fe(4)(HPBA)(2)(μ-CH(3)COO)(2)(μ-O)(μ-OH)(OH(2))(2)]ClO(4)·5H(2)O. The phosphoesterase-like activity of the complex in 1:1 acetonitrile/water has now been investigated using the substrate 2,4-bis(dinitrophenyl)phosphate. The pH dependence of the catalytic rate revealed a non-symmetric bell-shaped profile, with a finite but non-zero rate at high pH. Unlike the traditional approach usually employed to analyse these bell-shaped profiles, the approach used here involved incorporating additional species which contribute to the overall activity. Employing this approach, we show that the complex has a k (cat) of 1.6 (±0.2) × 10(-3) s(-1), three kinetically relevant pK (a) values of 5.3, 6.2 and 8.4, with K (M) of 7.4 ± 0.6 mM. The kinetic parameters are similar to those reported for heterovalent PAP biomimetics. Additionally, it is observed that, unlike the enzyme, the oxidation state is not the determining factor for catalytic activity.

Unique spectral signatures of the nucleic acid dye acridine orange can distinguish cell death by apoptosis and necroptosis

Publisher: Rockefeller University Press

Date: 06-03-2017

DOI: 10.1083/JCB.201602028

Abstract: Cellular injury and death are ubiquitous features of disease, yet tools to detect them are limited and insensitive to subtle pathological changes. Acridine orange (AO), a nucleic acid dye with unique spectral properties, enables real-time measurement of RNA and DNA as proxies for cell viability during exposure to various noxious stimuli. This tool illuminates spectral signatures unique to various modes of cell death, such as cells undergoing apoptosis versus necrosis/necroptosis. This new approach also shows that cellular RNA decreases during necrotic, necroptotic, and apoptotic cell death caused by demyelinating, ischemic, and traumatic injuries, implying its involvement in a wide spectrum of tissue pathologies. Furthermore, cells with pathologically low levels of cytoplasmic RNA are detected earlier and in higher numbers than with standard markers including TdT-mediated dUTP biotin nick-end labeling and cleaved caspase 3 immunofluorescence. Our technique highlights AO-labeled cytoplasmic RNA as an important early marker of cellular injury and a sensitive indicator of various modes of cell death in a range of experimental models.

Product release is rate-limiting for catalytic processing by the Dengue virus protease

Publisher: Springer Science and Business Media LLC

Date: 29-11-2016

DOI: 10.1038/SREP37539

Abstract: Dengue Virus (DENV) is the most prevalent global arbovirus, yet despite an increasing burden to health care there are currently no therapeutics available to treat infection. A potential target for antiviral drugs is the two-component viral protease NS2B-NS3pro, which is essential for viral replication. Interactions between the two components have been investigated here by probing the effect on the rate of enzyme catalysis of key mutations in a mobile loop within NS2B that is located at the interface of the two components. Steady-state kinetic assays indicated that the mutations greatly affect catalytic turnover. However, single turnover and fluorescence experiments have revealed that the mutations predominantly affect product release rather than substrate binding. Fluorescence analysis also indicated that the addition of substrate triggers a near-irreversible change in the enzyme conformation that activates the catalytic centre. Based on this mechanistic insight, we propose that residues within the mobile loop of NS2B control product release and present a new target for design of potent Dengue NS2B-NS3 protease inhibitors.

Rapid-Freeze-Quench Magnetic Circular Dichroism of Intermediate X in Ribonucleotide Reductase:  New Structural Insight

Publisher: American Chemical Society (ACS)

Date: 23-08-2003

DOI: 10.1021/JA036556E

Abstract: To elucidate the electronic structure of intermediate X in the oxygen activation reaction of the R2 subunit of ribonucleotide reductase, a protocol has been developed to perform magnetic circular dichroism (MCD) on a rapid-freeze-quench, strain free optical s le. RFQ-MCD data have been collected on intermediate X in the double mutant of R2, Y122/Y356F. While X has been reported to exhibit a broad absorption band at 365 nm, there are at least 10 electronic transitions observed at low-temperature MCD. From C0/D0 ratios, the transitions of X can be ided into three regions: 16 000-22 000 cm-1 region involving spin-allowed ligand field transitions of the Fe(IV), 23 000-24 000 cm-1 region of spin-forbidden, spin-flip transitions on the Fe(IV), and the charge transfer (CT) region from 26 000 to 32 000 cm-1. The C0/D0 ratios from d --> d and CT transitions strongly support significant Fe(IV) character coupled into the paramagnetic center. Ligand field (spin-allowed d --> d region) analysis allows the bis-mu-oxo and mu-oxo plus other monoanionic bridge possibilities for the structure of intermediate X to be distinguished, providing new insight into the molecular mechanism of the cluster formation in R2.

Purple acid phosphatases from bacteria: similarities to mammalian and plant enzymes

Publisher: Elsevier BV

Date: 09-2000

DOI: 10.1016/S0378-1119(00)00305-X

Abstract: Mammalian and plant purple acid phosphatases have similar active site structures despite low sequence identity (<20%). Although no bacterial enzyme has been purified, a sequence database search revealed that genes that could encode potential purple acid phosphatases may be restricted to a small number of organisms (i.e. myco- and cyanobacteria). Analysis of their deduced amino acid sequences and predicted secondary structures indicates that the cyanobacterial enzyme is similar to both the mammalian and the recently discovered low-molecular-weight plant purple acid phosphatases, while the mycobacterial enzyme is homologous to the fungal and high-molecular-weight plant purple acid phosphatases. Homology models indicate that both bacterial proteins appear to be similar to mammalian purple acid phosphatases in the immediate vicinity of the active site. It is likely that these enzymes act as Fenton-type catalysts in order to prevent damage caused by reactive oxygen species generated by invaded host cells (M. tuberculosis) or by the light-harvesting complex (Synechocystis sp.).

Formation of Catalytically Active Binuclear Center of Glycerophosphodiesterase: A Molecular Dynamics Study

Publisher: American Chemical Society (ACS)

Date: 03-05-2018

DOI: 10.1021/ACS.JPCB.8B02046

Abstract: Glycerophosphodiesterase (GpdQ) is a binuclear metallophosphatase that catalyzes the hydrolytic cleavage of mono-, di-, and triphosphoester bonds of a wide range of critical molecules. Upon substrate binding, this enzyme undergoes a complex transformation from an inactive mononuclear form (E

Comparative investigation of the reaction mechanisms of the organophosphate-degrading phosphotriesterases from Agrobacterium radiobacter (OpdA) and Pseudomonas diminuta (OPH)

Publisher: Springer Science and Business Media LLC

Date: 08-08-2014

DOI: 10.1007/S00775-014-1183-9

Abstract: Metal ion-dependent, organophosphate-degrading enzymes have acquired increasing attention due to their ability to degrade and thus detoxify commonly used pesticides and nerve agents such as sarin. The best characterized of these enzymes are from Pseudomonas diminuta (OPH) and Agrobacterium radiobacter (OpdA). Despite high sequence homology (>90 % identity) and conserved metal ion coordination these enzymes display considerable variations in substrate specificity, metal ion affinity reference and reaction mechanism. In this study, we highlight the significance of the presence (OpdA) or absence (OPH) of an extended hydrogen bond network in the active site of these enzymes for the modulation of their catalytic properties. In particular, the second coordination sphere residue in position 254 (Arg in OpdA, His in OPH) is identified as a crucial factor in modulating the substrate preference and binding of these enzymes. Inhibition studies with fluoride also support a mechanism for OpdA whereby the identity of the hydrolysis-initiating nucleophile changes as the pH is altered. The same is not observed for OPH.

Identification and preliminary characterization of novel B3-type metallo-β-lactamases

Publisher: Scientific Research Publishing, Inc.

Date: 2013

DOI: 10.4236/AJMB.2013.34026

Mapping the human genetic architecture of COVID-19

Publisher: Springer Science and Business Media LLC

Date: 08-07-2021

DOI: 10.1038/S41586-021-03767-X

Abstract: The genetic make-up of an in idual contributes to the susceptibility and response to viral infection. Although environmental, clinical and social factors have a role in the chance of exposure to SARS-CoV-2 and the severity of COVID-19 1,2 , host genetics may also be important. Identifying host-specific genetic factors may reveal biological mechanisms of therapeutic relevance and clarify causal relationships of modifiable environmental risk factors for SARS-CoV-2 infection and outcomes. We formed a global network of researchers to investigate the role of human genetics in SARS-CoV-2 infection and COVID-19 severity. Here we describe the results of three genome-wide association meta-analyses that consist of up to 49,562 patients with COVID-19 from 46 studies across 19 countries. We report 13 genome-wide significant loci that are associated with SARS-CoV-2 infection or severe manifestations of COVID-19. Several of these loci correspond to previously documented associations to lung or autoimmune and inflammatory diseases 3–7 . They also represent potentially actionable mechanisms in response to infection. Mendelian randomization analyses support a causal role for smoking and body-mass index for severe COVID-19 although not for type II diabetes. The identification of novel host genetic factors associated with COVID-19 was made possible by the community of human genetics researchers coming together to prioritize the sharing of data, results, resources and analytical frameworks. This working model of international collaboration underscores what is possible for future genetic discoveries in emerging pandemics, or indeed for any complex human disease.

Common, low-frequency, rare, and ultra-rare coding variants contribute to COVID-19 severity

Publisher: Springer Science and Business Media LLC

Date: 10-12-2021

DOI: 10.1007/S00439-021-02397-7

Abstract: The combined impact of common and rare exonic variants in COVID-19 host genetics is currently insufficiently understood. Here, common and rare variants from whole-exome sequencing data of about 4000 SARS-CoV-2-positive in iduals were used to define an interpretable machine-learning model for predicting COVID-19 severity. First, variants were converted into separate sets of Boolean features, depending on the absence or the presence of variants in each gene. An ensemble of LASSO logistic regression models was used to identify the most informative Boolean features with respect to the genetic bases of severity. The Boolean features selected by these logistic models were combined into an Integrated PolyGenic Score that offers a synthetic and interpretable index for describing the contribution of host genetics in COVID-19 severity, as demonstrated through testing in several independent cohorts. Selected features belong to ultra-rare, rare, low-frequency, and common variants, including those in linkage disequilibrium with known GWAS loci. Noteworthily, around one quarter of the selected genes are sex-specific. Pathway analysis of the selected genes associated with COVID-19 severity reflected the multi-organ nature of the disease. The proposed model might provide useful information for developing diagnostics and therapeutics, while also being able to guide bedside disease management.

Metabolomics of Non-Saccharomyces Yeasts in Fermented Beverages

Publisher: American Chemical Society (ACS)

Date: 07-2022

DOI: 10.26434/CHEMRXIV-2022-GF5DZ

Abstract: Fermented beverages have been consumed for millennia and today support a global industry producing erse products. Saccharomyces yeasts currently dominate the fermented beverage industry, but consumer demands for alternative products with a variety of sensory profiles and actual or perceived health benefits are driving the ersification and use of non-Saccharomyces yeasts. The ersity of flavours, aromas, and other sensory characteristics that can be obtained by using non-Saccharomyces yeasts in fermentation is, in large parts, due to the erse secondary metabolites they produce compared to conventional Saccharomyces yeast. Here, we review the use of metabolomic analyses of non- Saccharomyces yeasts to explore their impact on the sensory characteristics of fermented beverages. We highlight several key species currently used in the industry, including Brettanomyces, Torulaspora, Lachancea, and Saccharomycodes, and emphasize the future potential for the use of non- Saccharomyces yeasts in the production of erse fermented beverages.

Phosphate forms an unusual tripodal complex with the Fe–Mn center of sweet potato purple acid phosphatase

Publisher: Proceedings of the National Academy of Sciences

Date: 29-12-2004

DOI: 10.1073/PNAS.0407239102

Abstract: Purple acid phosphatases (PAPs) are a family of binuclear metalloenzymes that catalyze the hydrolysis of phosphoric acid esters and anhydrides. A PAP in sweet potato has a unique, strongly antiferromagnetically coupled Fe(III)–Mn(II) center and is distinguished from other PAPs by its increased catalytic efficiency for a range of activated and unactivated phosphate esters, its strict requirement for Mn(II), and the presence of a μ-oxo bridge at pH 4.90. This enzyme displays maximum catalytic efficiency ( k cat / K m ) at pH 4.5, whereas its catalytic rate constant ( k cat ) is maximal at near-neutral pH, and, in contrast to other PAPs, its catalytic parameters are not dependent on the pK a of the leaving group. The crystal structure of the phosphate-bound Fe(III)–Mn(II) PAP has been determined to 2.5-Å resolution (final R free value of 0.256). Structural comparisons of the active site of sweet potato, red kidney bean, and mammalian PAPs show several amino acid substitutions in the sweet potato enzyme that can account for its increased catalytic efficiency. The phosphate molecule binds in an unusual tripodal mode to the two metal ions, with two of the phosphate oxygen atoms binding to Fe(III) and Mn(II), a third oxygen atom bridging the two metal ions, and the fourth oxygen pointing toward the substrate binding pocket. This binding mode is unique among the known structures in this family but is reminiscent of phosphate binding to urease and of sulfate binding to λ protein phosphatase. The structure and kinetics support the hypothesis that the bridging oxygen atom initiates hydrolysis.

Anomalous scattering analysis of Agrobacterium radiobacter phosphotriesterase: the prominent role of iron in the heterobinuclear active site

Publisher: Portland Press Ltd.

Date: 13-07-2006

DOI: 10.1042/BJ20060276

Abstract: Bacterial phosphotriesterases are binuclear metalloproteins for which the catalytic mechanism has been studied with a variety of techniques, principally using active sites reconstituted in vitro from apoenzymes. Here, atomic absorption spectroscopy and anomalous X-ray scattering have been used to determine the identity of the metals incorporated into the active site in vivo. We have recombinantly expressed the phosphotriesterase from Agrobacterium radiobacter (OpdA) in Escherichia coli grown in medium supplemented with 1 mM CoCl2 and in unsupplemented medium. Anomalous scattering data, collected from a single crystal at the Fe–K, Co–K and Zn–K edges, indicate that iron and cobalt are the primary constituents of the two metal-binding sites in the catalytic centre (α and β) in the protein expressed in E. coli grown in supplemented medium. Comparison with OpdA expressed in unsupplemented medium demonstrates that the cobalt present in the supplemented medium replaced zinc at the β-position of the active site, which results in an increase in the catalytic efficiency of the enzyme. These results suggest an essential role for iron in the catalytic mechanism of bacterial phosphotriesterases, and that these phosphotriesterases are natively heterobinuclear iron–zinc enzymes.

Analogues of the Herbicide, N-Hydroxy-N-isopropyloxamate, Inhibit Mycobacterium tuberculosis Ketol-Acid Reductoisomerase and Their Prodrugs Are Promising Anti-TB Drug Leads

Publisher: American Chemical Society (ACS)

Date: 29-01-2021

DOI: 10.1021/ACS.JMEDCHEM.0C01919

Structure-activity relationship study and optimisation of 2-aminopyrrole-1-benzyl-4,5-diphenyl-1 H -pyrrole-3-carbonitrile as a broad spectrum metallo-β-lactamase inhibitor

Publisher: Elsevier BV

Date: 09-2017

DOI: 10.1016/J.EJMECH.2017.05.061

Abstract: A SAR study on derivatives of 2-amino-1-benzyl-4,5-diphenyl-1H-pyrrole-3-carbonitrile 5a revealed that the 3-carbonitrile group, vicinal 4,5-diphenyl and N-benzyl side chains of the pyrrole are important for the inhibitory potencies of these compounds against members representing the three main subclasses of metallo-β-lactamases (MBLs), i.e. IMP-1 (representing the B1 subgroup), CphA (B2) and AIM-1 (B3). Coupling of 5a with a series of acyl chlorides and anhydrides led to the discovery of two N-acylamide derivatives, 10 and 11, as the two most potent IMP-1 inhibitors in this series. However, these compounds are less effective towards CphA and AIM-1. The N-benzoyl derivative of 5a retained potent in vitro activity against each of MBLs tested (with inhibition constants in the low μM range). Importantly, this compound also significantly enhanced the sensitivity of IMP-1, CphA- or AIM-1-producing cell cultures towards meropenem. This compound presents a promising starting point for the development of a universal MBL inhibitor, targeting members of each of the major subgroups of this family of enzymes.

Monoesterase Activity of a Purple Acid Phosphatase Mimic with a Cyclam Platform

Publisher: Wiley

Date: 10-01-2012

DOI: 10.1002/CHEM.201100229

Abstract: The synthesis and characterization of a novel dinucleating ligand L (L=4,11-dimethyl-1,8-bis{2-[N-(di-2-pyridylmethyl)amino]ethyl}cyclam) and its μ-oxo-bridged diferric complex [(H(2)L){Fe(III)(2)(O)}(Cl)(4)](2+) are reported. This diiron(III) complex is the first ex le of a truly functional purple acid phosphatase (PAP) mimic as it accelerates the hydrolysis of the activated phosphomonoester 2,4-dinitrophenyl phosphate (DNPP). The spectroscopic and kinetic data indicate that only substrates that are monodentately bound to one of the two ferric ions can be attacked by a suitable nucleophile. This is, most probably, a terminal iron(III)-bound hydroxide. DFT calculations support this assumption and also highlight the importance of secondary interactions, exerted by the protonated cyclam platform, for the positioning and activation of the iron(III)-bound substrate. Similar effects are postulated in the native enzyme but addressed in PAP mimics for the first time.

Improving a Natural Enzyme Activity through Incorporation of Unnatural Amino Acids

Publisher: American Chemical Society (ACS)

Date: 16-12-2011

DOI: 10.1021/JA106416G

Abstract: The bacterial phosphotriesterases catalyze hydrolysis of the pesticide paraoxon with very fast turnover rates and are thought to be near to their evolutionary limit for this activity. To test whether the naturally evolved turnover rate could be improved through the incorporation of unnatural amino acids and to probe the role of peripheral active site residues in nonchemical steps of the catalytic cycle (substrate binding and product release), we replaced the naturally occurring tyrosine amino acid at position 309 with unnatural L-(7-hydroxycoumarin-4-yl)ethylglycine (Hco) and L-(7-methylcoumarin-4-yl)ethylglycine amino acids, as well as leucine, phenylalanine, and tryptophan. Kinetic analysis suggests that the 7-hydroxyl group of Hco, particularly in its deprotonated state, contributes to an increase in the rate-limiting product release step of substrate turnover as a result of its electrostatic repulsion of the negatively charged 4-nitrophenolate product of paraoxon hydrolysis. The 8-11-fold improvement of this already highly efficient catalyst through a single rationally designed mutation using an unnatural amino acid stands in contrast to the difficulty in improving this native activity through screening hundreds of thousands of mutants with natural amino acids. These results demonstrate that designer amino acids provide easy access to new and valuable sequence and functional space for the engineering and evolution of existing enzyme functions.

The reaction mechanism of the Ga(III)Zn(II) derivative of uteroferrin and corresponding biomimetics

Publisher: Springer Science and Business Media LLC

Date: 15-08-2007

DOI: 10.1007/S00775-007-0286-Y

Abstract: Purple acid phosphatase from pig uterine fluid (uteroferrin), a representative of the erse family of binuclear metallohydrolases, requires a heterovalent Fe(III)Fe(II) center for catalytic activity. The active-site structure and reaction mechanism of this enzyme were probed with a combination of methods including metal ion replacement and biomimetic studies. Specifically, the asymmetric ligand 2-bis{[(2-pyridylmethyl)-aminomethyl]-6-[(2-hydroxybenzyl)(2-pyridylmethyl)]aminomethyl}-4-methylphenol and two symmetric analogues that contain the softer and harder sites of the asymmetric unit were employed to assess the site selectivity of the trivalent and alent metal ions using (71)Ga NMR, mass spectrometry and X-ray crystallography. An exclusive preference of the harder site of the asymmetric ligand for the trivalent metal ion was observed. Comparison of the reactivities of the biomimetics with Ga(III)Zn(II) and Fe(III)Zn(II) centers indicates a higher turnover for the former, suggesting that the M(III)-bound hydroxide acts as the reaction-initiating nucleophile. Catalytically active Ga(III)Zn(II) and Fe(III)Zn(II) derivatives were also generated in the active site of uteroferrin. As in the case of the biomimetics, the Ga(III) derivative has increased reactivity, and a comparison of the pH dependence of the catalytic parameters of native uteroferrin and its metal ion derivatives supports a flexible mechanistic strategy whereby both the mu-(hydr)oxide and the terminal M(III)-bound hydroxide can act as nucleophiles, depending on the metal ion composition, the geometry of the second coordination sphere and the substrate.

A New Mixed-Valence Mn(II)Mn(III) Compound With Catalase and Superoxide Dismutase Activities

Publisher: Frontiers Media SA

Date: 05-11-2018

DOI: 10.3389/FCHEM.2018.00491

Editorial: Advances in the Development of Artificial Metalloenzymes

Publisher: Frontiers Media SA

Date: 28-08-2019

DOI: 10.3389/FCHEM.2019.00599

Recent Advances in Heterogeneous Catalytic Systems Containing Metal Ions for Phosphate Ester Hydrolysis

Publisher: Wiley

Date: 23-09-2020

DOI: 10.1002/CHEM.202002333

Advances in the Development of Artificial Metalloenzymes

Publisher: Frontiers Media SA

Date: 2019

DOI: 10.3389/978-2-88963-153-7

Structural and spectroscopic studies of a model for catechol oxidase

Publisher: Springer Science and Business Media LLC

Date: 10-01-2008

DOI: 10.1007/S00775-007-0334-7

Abstract: A binuclear copper complex, [Cu2(BPMP) (OAc)2][ClO4] x H2O, has been prepared using the binucleating ligand 2,6-bis[bis(pyridin-2-ylmethylamino)methyl]-4-methylphenol (H-BPMP). The X-ray crystal structure reveals the copper centers to have a five-coordinate square pyramidal geometry, with the acetate ligands bound terminally. The bridging phenolate occupies the apical position of the square-based pyramids and magnetic susceptibility, electron paramagnetic resonance (EPR) and variable-temperature variable-field magnetic circular dichroism (MCD) measurements indicate that the two centers are very weakly antiferromagnetically coupled (J = -0.6 cm(-1)). Simulation of the dipole-dipole-coupled EPR spectrum showed that in solution the Cu-O-Cu angle was increased from 126 degrees to 160 degrees and that the internuclear distance was larger than that observed crystallographically. The high-resolution spectroscopic information obtained has been correlated with a detailed ligand-field analysis to gain insight into the electronic structure of the complex. Symmetry arguments have been used to demonstrate that the sign of the MCD is characteristic of the tetragonally elongated environment. The complex also displays catecholase activity (k(cat) = 15 +/- 1.5 min(-1), K(M) = 6.4 +/- 1.8 mM), which is compared with other dicopper catechol oxidase models.

Infection Control Measures and Prevalence of SARS-CoV-2 IgG among 4,554 University Hospital Employees, Munich, Germany

Publisher: Centers for Disease Control and Prevention (CDC)

Date: 03-2022

DOI: 10.3201/EID2803.204436

Abstract: Hospital staff are at high risk for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection during the coronavirus disease (COVID-19) pandemic. This cross-sectional study aimed to determine the prevalence of SARS-CoV-2 infection in hospital staff at the University Hospital rechts der Isar in Munich, Germany, and identify modulating factors. Overall seroprevalence of SARS-CoV-2-IgG in 4,554 participants was 2.4%. Staff engaged in direct patient care, including those working in COVID-19 units, had a similar probability of being seropositive as non-patient-facing staff. Increased probability of infection was observed in staff reporting interactions with SARS-CoV-2‒infected coworkers or private contacts or exposure to COVID-19 patients without appropriate personal protective equipment. Analysis of spatiotemporal trajectories identified that distinct hotspots for SARS-CoV-2‒positive staff and patients only partially overlap. Patient-facing work in a healthcare facility during the SARS-CoV-2 pandemic might be safe as long as adequate personal protective equipment is used and infection prevention practices are followed inside and outside the hospital.

Product Profiles of Promiscuous Enzymes Can be Altered by Controlling In Vivo Spatial Organization

Publisher: Wiley

Date: 26-09-2023

DOI: 10.1002/ADVS.202303415

The Catalytic Mechanisms of Binuclear Metallohydrolases

Publisher: American Chemical Society (ACS)

Date: 06-2006

DOI: 10.1021/CR050318F

Malonate-bound structure of the glycerophosphodiesterase from Enterobacter aerogenes (GpdQ) and characterization of the native Fe2+ metal-ion preference

Publisher: International Union of Crystallography (IUCr)

Date: 05-07-2008

DOI: 10.1107/S1744309108017600

β-Lactam antibiotic-degrading enzymes from non-pathogenic marine organisms: a potential threat to human health

Publisher: Springer Science and Business Media LLC

Date: 14-03-2015

DOI: 10.1007/S00775-015-1250-X

Abstract: Metallo-β-lactamases (MBLs) are a family of Zn(II)-dependent enzymes that inactivate most of the commonly used β-lactam antibiotics. They have emerged as a major threat to global healthcare. Recently, we identified two novel MBL-like proteins, Maynooth IMipenemase-1 (MIM-1) and Maynooth IMipenemase-2 (MIM-2), in the marine organisms Novosphingobium pentaromativorans and Simiduia agarivorans, respectively. Here, we demonstrate that MIM-1 and MIM-2 have catalytic activities comparable to those of known MBLs, but from the pH dependence of their catalytic parameters it is evident that both enzymes differ with respect to their mechanisms, with MIM-1 preferring alkaline and MIM-2 acidic conditions. Both enzymes require Zn(II) but activity can also be reconstituted with other metal ions including Co(II), Mn(II), Cu(II) and Ca(II). Importantly, the substrate preference of MIM-1 and MIM-2 appears to be influenced by their metal ion composition. Since neither N. pentaromativorans nor S. agarivorans are human pathogens, the precise biological role(s) of MIM-1 and MIM-2 remains to be established. However, due to the similarity of at least some of their in vitro functional properties to those of known MBLs, MIM-1 and MIM-2 may provide essential structural insight that may guide the design of as of yet elusive clinically useful MBL inhibitors.

Efficient Green Light Acclimation of the Green Algae Picochlorum sp. Triggering Geranylgeranylated Chlorophylls

Publisher: Frontiers Media SA

Date: 28-04-2022

DOI: 10.3389/FBIOE.2022.885977

Abstract: In analogy to higher plants, eukaryotic microalgae are thought to be incapable of utilizing green light for growth, due to the “green gap” in the absorbance profiles of their photosynthetic pigments. This study demonstrates, that the marine chlorophyte Picochlorum sp . is able to grow efficiently under green light emitting diode (LED) illumination. Picochlorum sp. growth and pigment profiles under blue, red, green and white LED illumination (light intensity: 50–200 μmol m −2 s −1 ) in bottom-lightened shake flask cultures were evaluated. Green light-treated cultures showed a prolonged initial growth lag phase of one to 2 days, which was subsequently compensated to obtain comparable biomass yields to red and white light controls (approx. 0.8 g DW L −1 ). Interestingly, growth and final biomass yields of the green light-treated s le were higher than under blue light with equivalent illumination energies. Further, pigment analysis indicated, that during green light illumination, Picochlorum sp . formed unknown pigments (X1-X4). Pigment concentrations increased with illumination intensity and were most abundant during the exponential growth phase. Mass spectrometry and nuclear magnetic resonance data indicated, that pigments X1-X2 and X3-X4 are derivatives of chlorophyll b and a , which harbor C=C bonds in the phytol side chain similar to geranylgeranylated chlorophylls. Thus, for the first time, the natural accumulation of large pools (approx. 12 mg g DW −1 ) of chlorophyll intermediates with incomplete hydrogenation of their phytyl chains is demonstrated for algae under monochromatic green light (Peak λ 510 nm, full width at half maximum 91 nm). The ability to utilize green light offers competitive advantages for enhancing biomass production, particularly under conditions of dense cultures, long light pathways and high light intensity. Green light acclimation for an eukaryotic microalgae in conjunction with the formation of new aberrant geranylgeranylated chlorophylls and high efficiency of growth rates are novel for eukaryotic microalgae. Illumination with green light could enhance productivity in industrial processes and trigger the formation of new metabolites–thus, underlying mechanisms require further investigation.

Determination of the catalytic activity of binuclear metallohydrolases using isothermal titration calorimetry

Publisher: Springer Science and Business Media LLC

Date: 12-01-2014

DOI: 10.1007/S00775-013-1079-0

Abstract: Binuclear metallohydrolases are a large and erse family of enzymes that are involved in numerous metabolic functions. An increasing number of members find applications as drug targets or in processes such as bioremediation. It is thus essential to have an assay available that allows the rapid and reliable determination of relevant catalytic parameters (k cat, K m, and k cat/K m). Continuous spectroscopic assays are frequently only possible by using synthetic (i.e., nonbiological) substrates that possess a suitable chromophoric marker (e.g., nitrophenol). Isothermal titration calorimetry, in contrast, affords a rapid assay independent of the chromophoric properties of the substrate-the heat associated with the hydrolytic reaction can be directly related to catalytic properties. Here, we demonstrate the efficiency of the method on several selected ex les of this family of enzymes and show that, in general, the catalytic parameters obtained by isothermal titration calorimetry are in good agreement with those obtained from spectroscopic assays.

Broad spectrum antibiotic-degrading metallo-beta-lactamases are phylogenetically diverse

Publisher: Oxford University Press (OUP)

Date: 15-06-2020

DOI: 10.1007/S13238-020-00736-4

Metallo‐β‐lactamases and Their Biomimetic Complexes

Publisher: Wiley

Date: 06-2014

DOI: 10.1002/EJIC.201402203

Dinuclear copper(II) complexes with derivative triazine ligands as biomimetic models for catechol oxidases and nucleases

Publisher: Elsevier BV

Date: 12-2020

DOI: 10.1016/J.JINORGBIO.2020.111249

Binuclear Metallohydrolases: Complex Mechanistic Strategies for a Simple Chemical Reaction

Publisher: American Chemical Society (ACS)

Date: 14-06-2012

DOI: 10.1021/AR300067G

Abstract: Binuclear metallohydrolases are a large family of enzymes that require two closely spaced transition metal ions to carry out a plethora of hydrolytic reactions. Representatives include purple acid phosphatases (PAPs), enzymes that play a role in bone metabolism and are the only member of this family with a heterovalent binuclear center in the active form (Fe(3+)-M(2+), M = Fe, Zn, Mn). Other members of this family are urease, which contains a di-Ni(2+) center and catalyzes the breakdown of urea, arginase, which contains a di-Mn(2+) center and catalyzes the final step in the urea cycle, and the metallo-β-lactamases, which contain a di-Zn(2+) center and are virulence factors contributing to the spread of antibiotic-resistant pathogens. Binuclear metallohydrolases catalyze numerous vital reactions and are potential targets of drugs against a wide variety of human disorders including osteoporosis, various cancers, antibiotic resistance, and erectile dysfunctions. These enzymes also tend to catalyze more than one reaction. An ex le is an organophosphate (OP)-degrading enzyme from Enterobacter aerogenes (GpdQ). Although GpdQ is part of a pathway that is used by bacteria to degrade glycerolphosphoesters, it hydrolyzes a variety of other phosphodiesters and displays low levels of activity against phosphomono- and triesters. Such a promiscuous nature may have assisted the apparent recent evolution of some binuclear metallohydrolases to deal with situations created by human intervention such as OP pesticides in the environment. OP pesticides were first used approximately 70 years ago, and therefore the enzymes that bacteria use to degrade them must have evolved very quickly on the evolutionary time scale. The promiscuous nature of enzymes such as GpdQ makes them ideal candidates for the application of directed evolution to produce new enzymes that can be used in bioremediation and against chemical warfare. In this Account, we review the mechanisms employed by binuclear metallohydrolases and use PAP, the OP-degrading enzyme from Agrobacterium radiobacter (OPDA), and GpdQ as representative systems because they illustrate both the ersity and similarity of the reactions catalyzed by this family of enzymes. The majority of binuclear metallohydrolases utilize metal ion-activated water molecules as nucleophiles to initiate hydrolysis, while some, such as alkaline phosphatase, employ an intrinsic polar amino acid. Here we only focus on catalytic strategies applied by the former group.

Enabling the Direct Enzymatic Dehydration of d-Glycerate to Pyruvate as the Key Step in Synthetic Enzyme Cascades Used in the Cell-Free Production of Fine Chemicals

Publisher: American Chemical Society (ACS)

Date: 03-02-2020

DOI: 10.1021/ACSCATAL.9B05068

Synthesis and kinetic testing of new inhibitors for a metallo-β-lactamase from Klebsiella pneumonia and Pseudomonas aeruginosa

Publisher: Elsevier BV

Date: 12-2011

DOI: 10.1016/J.EJMECH.2011.10.030

Abstract: There are currently no clinically useful inhibitors against metallo-β-lactamases (MBLs), enzymes that confer resistance against a broad spectrum of commonly used antibiotics and that are produced by an increasing number of bacterial pathogens. New pyrrole derivatives were synthesized and assayed for their inhibitory effect on the catalytic activity of the IMP-1 MBL from Pseudomonas aeruginosa and Klebsiella pneumoniae. Six compounds tested (3a-3c, 5, 7 and 8) show micromolar inhibition constants (K(i) values range from ∼10 to 30 μM). In silico docking was employed to investigate the binding mode of the strongest inhibitor, 3b, in the active site of IMP-1. Implications for further improvements of binding efficiency and specificity are discussed.

Enhancing the Thermal and Kinetic Stability of Ketol-Acid Reductoisomerase, a Central Catalyst of a Cell-Free Enzyme Cascade for the Manufacture of Platform Chemicals

Publisher: MDPI AG

Date: 10-08-2022

DOI: 10.3390/APPLBIOSCI1020011

Abstract: The branched-chain amino acids (BCAAs) leucine, isoleucine and valine are synthesized via a common biosynthetic pathway. Ketol-acid reductoisomerase (KARI) is the second enzyme in this pathway. In addition to its role in BCAA biosynthesis, KARI catalyzes two rate-limiting steps that are key components of a cell-free biofuel biosynthesis route. For industrial applications, reaction temperature and enzyme stability are key factors that affect process robustness and product yield. Here, we have solved the cryo-EM structure (2.94 Å resolution) of a homododecameric Class I KARI (from C ylobacter jejuni) and demonstrated how a triad of amino acid side chains plays a crucial role in promoting the oligomerization of this enzyme. Importantly, both its thermal and solvent stability are greatly enhanced in the dodecameric state when compared to its dimeric counterpart (apparent melting temperatures (Tm) of 83.1 °C and 51.5 °C, respectively). We also employed protein design (PROSS) for a tetrameric Class II KARI (from Escherichia coli) to generate a variant with improved thermal and solvent stabilities. In total, 34 mutations were introduced, which did not affect the oligomeric state of this enzyme but resulted in a fully functional catalyst with a significantly elevated Tm (58.5 °C vs. 47.9 °C for the native version).

Structural and Functional Insight into the Mechanism of the Fe−S Cluster‐Dependent Dehydratase from Paralcaligenes ureilyticus

Publisher: Wiley

Date: 27-12-2022

DOI: 10.1002/CHEM.202203140

Abstract: Enzyme‐catalyzed reaction cascades play an increasingly important role for the sustainable manufacture of erse chemicals from renewable feedstocks. For instance, dehydratases from the ilvD/EDD superfamily have been embedded into a cascade to convert glucose via pyruvate to isobutanol, a platform chemical for the production of aviation fuels and other valuable materials. These dehydratases depend on the presence of both a Fe−S cluster and a alent metal ion for their function. However, they also represent the rate‐limiting step in the cascade. Here, catalytic parameters and the crystal structure of the dehydratase from Paralcaligenes ureilyticus ( Pu DHT, both in presence of Mg 2+ and Mn 2+ ) were investigated. Rate measurements demonstrate that the presence of stoichiometric concentrations Mn 2+ promotes higher activity than Mg 2+ , but at high concentrations the former inhibits the activity of Pu DHT. Molecular dynamics simulations identify the position of a second binding site for the alent metal ion. Only binding of Mn 2+ (not Mg 2+ ) to this site affects the ligand environment of the catalytically essential alent metal binding site, thus providing insight into an inhibitory mechanism of Mn 2+ at higher concentrations. Furthermore, in silico docking identified residues that play a role in determining substrate binding and selectivity. The combined data inform engineering approaches to design an optimal dehydratase for the cascade.

Spectroscopic and mechanistic studies of dinuclear metallohydrolases and their biomimetic complexes

Publisher: Royal Society of Chemistry (RSC)

Date: 2014

DOI: 10.1039/C3DT52287C

Abstract: An enhanced understanding of the metal ion binding and active site structural features of phosphoesterases such as the glycerophosphodiesterase from Enterobacter aerogenes (GpdQ), and the organophosphate degrading agent from Agrobacterium radiobacter (OpdA) have important consequences for potential applications. Coupled with investigations of the metalloenzymes, programs of study to synthesise and characterise model complexes based on these metalloenzymes can add to our understanding of structure and function of the enzymes themselves. This review summarises some of our work and illustrates the significance and contributions of model studies to knowledge in the area.

Electronic Structure Analysis of the Dinuclear Metal Center in the Bioremediator Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes

Publisher: American Chemical Society (ACS)

Date: 17-02-2010

DOI: 10.1021/IC901950C

Abstract: The glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a promiscuous, dinuclear metallohydrolase that has potential application in the remediation of organophosphate nerve agents and pesticides. GpdQ employs an unusual reaction mechanism in which the enzyme is predominantly mononuclear in the resting state, and substrate binding induces the formation of the catalytically competent dinuclear center (Hadler et al. J. Am. Chem. Soc. 2008, 130, 14129). Reactivity is further modulated by the coordination flexibility of Asn80, a ligand that binds to the second, loosely bound metal ion (Hadler et al. J. Am. Chem. Soc. 2009, 131, 11900). It is proposed that hydrolysis is initiated by a terminal, metal-bound hydroxide molecule which is activated at unusually low pH by electrostatic/hydrogen bonding interactions with a bridging hydroxide species. In this study, electronic structure analysis of the dinuclear center is employed to study the coordination environment of the dinuclear center at the resting and product-bound stage of catalysis. This is achieved through the use of variable temperature, variable field magnetic circular dichroism experiments involving the Co(II)-substituted wild type enzyme and its Asn80Asp variant. The data support the above model for the catalytic mechanism whereby the metal ion-bridging hydroxide molecule activates a terminally bound hydroxide nucleophile. Replacement of Asn80 by an aspartate residue does prevent coordination flexibility but also leads to cleavage of the mu-hydroxide bridge and reduced reactivity. This is the first study to investigate the electronic structure of an enzyme with a mu-1,1-carboxylate bridged dicobalt(II) center.

Comparison between the Geometric and Electronic Structures and Reactivities of {FeNO}⁷and {FeO₂}⁸Complexes: A Density Functional Theory Study

Publisher: American Chemical Society (ACS)

Date: 18-12-2003

DOI: 10.1021/JA036715U

Abstract: In a previous study, we analyzed the electronic structure of S = 3/2 [FeNO](7) model complexes [Brown et al. J. Am. Chem. Soc. 1995, 117, 715-732]. The combined spectroscopic data and SCF-X alpha-SW electronic structure calculations are best described in terms of Fe(III) (S = 5/2) antiferromagnetically coupled to NO(-) (S = 1). Many nitrosyl derivatives of non-heme iron enzymes have spectroscopic properties similar to those of these model complexes. These NO derivatives can serve as stable analogues of highly labile oxygen intermediates. It is thus essential to establish a reliable density functional theory (DFT) methodology for the geometry and energetics of [FeNO](7) complexes, based on detailed experimental data. This methodology can then be extended to the study of [FeO(2)](8) complexes, followed by investigations into the reaction mechanisms of non-heme iron enzymes. Here, we have used the model complex Fe(Me(3)TACN)(NO)(N(3))(2) as an experimental marker and determined that a pure density functional BP86 with 10% hybrid character and a mixed triple-zeta/double-zeta basis set lead to agreement between experimental and computational data. This methodology is then applied to optimize the hypothetical Fe(Me(3)TACN)(O(2))(N(3))(2) complex, where the NO moiety is replaced by O(2). The main geometric differences are an elongated Fe[bond]O(2) and a steeper Fe[bond]O[bond]O angle in the [FeO(2)](8) complex. The electronic structure of [FeO(2)](8) corresponds to Fe(III) (S = 5/2) antiferromagnetically coupled to O(2)(-) (S = 1/2), and, consistent with the extended bond length, the [FeO(2)](8) unit has only one Fe(III)-O(2)(-) bonding interaction, while the [FeNO](7) unit has both sigma and pi type Fe(III)-NO(-) bonds. This is in agreement with experiment as NO forms a more stable Fe(III)-NO(-) adduct relative to O(2)(-). Although NO is, in fact, harder to reduce, the resultant NO(-) species forms a more stable bond to Fe(III) relative to O(2)(-) due to the different bonding interactions.

Use of magnetic circular dichroism to study dinuclear metallohydrolases and the corresponding biomimetics

Publisher: Springer Science and Business Media LLC

Date: 07-2015

DOI: 10.1007/S00249-015-1053-6

Abstract: Magnetic circular dichroism (MCD) is a convenient technique for providing structural and mechanistic insight into enzymatic systems in solution. The focus of this review is on aspects of geometric and electronic structure that can be determined by MCD, and how this method can further our understanding of enzymatic mechanisms. Dinuclear Co(II) systems that catalyse hydrolytic reactions were selected to illustrate the approach. These systems all contain active sites with similar structures consisting of two Co(II) ions bridged by one or two carboxylates and a water or hydroxide. In most of these active sites one Co(II) is five-coordinate and one is six-coordinate, with differing binding affinities. It is shown how MCD can be used to determine which binding site--five or six-coordinate--has the greater affinity. Importantly, zero-field-splitting data and magnetic exchange coupling constants may be determined from the temperature and field dependence of MCD data. The relevance of these data to the function of the enzymatic systems is discussed.

Cover Picture: Total Synthesis and Complete Stereostructure of a Marine Macrolide Glycoside, (−)‐Lyngbyaloside B (Chem. Eur. J. 20/2016)

Publisher: Wiley

Date: 18-04-2016

DOI: 10.1002/CHEM.201601249

Inteins—A Focus on the Biotechnological Applications of Splicing-Promoting Proteins

Publisher: Scientific Research Publishing, Inc.

Date: 2015

DOI: 10.4236/AJMB.2015.52005

Phosphotyrosyl peptides and analogues as substrates and inhibitors of purple acid phosphatases

Publisher: Elsevier BV

Date: 04-2004

DOI: 10.1016/J.ABB.2004.01.008

Spectroscopic Characterization of the Active Fe^IIIFe^III and Fe^IIIFe^II Forms of a Purple Acid Phosphatase Model System

Publisher: American Chemical Society (ACS)

Date: 30-10-2012

DOI: 10.1021/IC301347T

Abstract: Two new dinucleating ligands (H3L(2) and HL(3)), derivatives of a well-known dinucleating ligand (HL(1)) with two bis-picolylamine sites connected to a bridging phenolate, with hydrogen-bonding donor groups at two of the pyridine moieties were designed and synthesized. Design of these ligands suggests that they will lead to dinuclear complexes with potential to stabilize phosphoester substrates as monodentate rather than bridging ligands. We report the diferric complexes [Fe(III)2(H2L(2))(OH)](4+) and [Fe(III)2(L(3))(OH)(OH2)2](4+), which have been characterized by spectrophotometric titrations, UV-vis, IR, NMR, EPR, and Mössbauer spectroscopy. The phosphatase activity of the diferric systems, in addition to the partially reduced heterovalent [Fe(III)Fe(II)(L(3))(OH)(OH2)2](3+) complex, has been investigated, and the complexes are shown to catalytically hydrolyze the activated phosphodiester substrate BDNPP (bis-dinitrophenylphosphate) as well as the corresponding phosphomonoester substrate DNPP (dinitrophenylphosphate). The results indicate that indeed the secondary interactions lead to an increase of the phosphatase activity and to active phosphomonoesterase catalysts. Interestingly, the heterovalent form of the HL(3)-based complex is more efficient than the diferric complex, and this is also discussed.

Synthesis, modelling and kinetic assays of potent inhibitors of purple acid phosphatase

Publisher: Elsevier BV

Date: 05-2011

DOI: 10.1016/J.BMCL.2011.03.024

Abstract: Purple acid phosphatases (PAPs) are binuclear metallohydrolases that have been isolated from various mammals, plants, fungi and bacteria. In mammals PAP activity is associated with bone resorption and can lead to bone metabolic disorders such as osteoporosis thus human PAP is an attractive target to develop anti-osteoporotic drugs. Based on a previous lead compound and rational drug design, acyl derivatives of α-aminonaphthylmethylphosphonic acid were synthesised and tested as PAP inhibitors. Kinetic analysis showed that they are good PAP inhibitors whose potencies improve with increasing acyl chain length. Maximum potency is reached when the number of carbons in the acyl chain is between 12 and 14. The most potent inhibitor of red kidney bean PAP is the dodecyl-derivative with K(ic)=5 μM, while the most potent pig PAP inhibitor is the tetradecyl-derivative with K(ic)=8 μM, the most potent inhibitor of a mammalian PAP yet reported.

A Heterodinuclear Fe^IIIZn^II Complex as a Mimic for Purple Acid Phosphatase with Site‐Specific Zn^II Binding

Publisher: Wiley

Date: 09-06-2015

DOI: 10.1002/EJIC.201500351

Captopril analogues as metallo-β-lactamase inhibitors

Publisher: Elsevier BV

Date: 03-2016

DOI: 10.1016/J.BMCL.2016.02.007

Abstract: A number of captopril analogues were synthesised and tested as inhibitors of the metallo-β-lactamase IMP-1. Structure-activity studies showed that the methyl group was unimportant for activity, and that the potencies of these inhibitors could be best improved by shortening the length of the mercaptoalkanoyl side-chain. Replacing the thiol group with a carboxylic acid led to complete loss of activity, and extending the length of the carboxylate group led to decreased potency. Good activity could be maintained by substituting the proline ring with pipecolic acid.

Insight on the interaction of an agmatinase-like protein with Mn2+ activator ions

Publisher: Elsevier BV

Date: 04-0003

DOI: 10.1016/J.JINORGBIO.2015.01.008

Abstract: Agmatinase is an enzyme that catalyzes the hydrolysis of agmatine, a compound that is associated with numerous functions in the brain of mammalian organisms such as neurotransmitter, anticonvulsant, antinociceptive, anxiolytic and antidepressant-like actions. To date the only characterized agmatinases with significant enzymatic activity were extracted from bacterial organisms. These agmatinases are closely related to another ureahydrolase, arginase both have binuclear Mn(2+) centers in their active sites. An agmatinase-like protein (ALP) from rat brain was identified that bears no sequence homology to known agmatinases (E. Uribe, M. Salas, S. Enriquez, M.S. Orellana, N. Carvajal, Arch. Biochem. Biophys. 461(2007) 146-150). Since all known ureahydrolases contain histidines in their binuclear Mn(2+) site each of the five histidine residues in ALP was in idually replaced by alanines to identify those that may be involved in metal ion binding. Reactivation assays and thermal stability measurements indicated that His206 is likely to interact with a Mn(2+) bound to a high affinity site. In contrast, His65 and possibly His435 are important for binding of a second Mn(2+) to a lower affinity site. Metal ion binding to that site is not only leading to an increase in reactivity but also enzyme stability. Thus, similar to bacterial agmatinases and some of the antibiotic-degrading, Zn(2+)-dependent metallo-β-lactamases ALP appears to be active in the mono and binuclear form, with binding of the second metal ion increasing both reactivity and stability.

Mammalian agmatinases constitute unusual members in the family of Mn 2+ -dependent ureahydrolases

Publisher: Elsevier BV

Date: 2017

DOI: 10.1016/J.JINORGBIO.2016.11.015

Abstract: Agmatine (1-amino-4-guanidinobutane) plays an important role in a range of metabolic functions, in particular in the brain. Agmatinases (AGMs) are enzymes capable of converting agmatine to the polyamine putrescine and urea. AGMs belong to the family of Mn

Sequence- and structure-guided improvement of the catalytic performance of a GH11 family xylanase from Bacillus subtilis

Publisher: Elsevier BV

Date: 11-2021

DOI: 10.1016/J.JBC.2021.101262

Metal Ions Play an Essential Catalytic Role in the Mechanism of Ketol–Acid Reductoisomerase

Publisher: Wiley

Date: 02-05-2016

DOI: 10.1002/CHEM.201600620

Abstract: Ketol–acid reductoisomerase (KARI) is a Mg 2+ ‐dependent enzyme in the branched‐chain amino acid biosynthesis pathway. It catalyses a complex two‐part reaction: an alkyl migration followed by a NADPH‐dependent reduction. Both reactions occur within the one active site, but in particular, the mechanism of the isomerisation step is poorly understood. Here, using a combination of kinetic, thermodynamic and spectroscopic techniques, the reaction mechanisms of both Escherichia coli and rice KARI have been investigated. We propose a conserved mechanism of catalysis, whereby a hydroxide, bridging the two Mg 2+ ions in the active site, initiates the reaction by abstracting a proton from the C2 alcohol group of the substrate. While the μ‐hydroxide‐bridged dimetallic centre is pre‐assembled in the bacterial enzyme, in plant KARI substrate binding leads to a reduction of the metal–metal distance with the concomitant formation of a hydroxide bridge. Only Mg 2+ is capable of promoting the isomerisation reaction, likely to be due to non‐competent substrate binding in the presence of other metal ions.

Kinetic and Spectroscopic Studies of N694C Lipoxygenase:  A Probe of the Substrate Activation Mechanism of a Nonheme Ferric Enzyme

Publisher: American Chemical Society (ACS)

Date: 25-05-2007

DOI: 10.1021/JA068503D

Purple acid phosphatase inhibitors as leads for osteoporosis chemotherapeutics

Publisher: Elsevier BV

Date: 09-2018

DOI: 10.1016/J.EJMECH.2018.08.004

Abstract: Purple acid phosphatases (PAPs) are metalloenzymes that catalyse the hydrolysis of phosphate esters under acidic conditions. Their active site contains a Fe(III)Fe(II) metal centre in mammals and a Fe(III)Zn(II) or Fe(III)Mn(II) metal centre in plants. In humans, elevated PAP levels in serum strongly correlate with the progression of osteoporosis and metabolic bone malignancies, which make PAP a target suitable for the development of chemotherapeutics to combat bone ailments. Due to difficulties in obtaining the human enzyme, the corresponding enzymes from red kidney bean and pig have been used previously to develop specific PAP inhibitors. Here, existing lead compounds were further elaborated to create a series of inhibitors with K

Inhibitors for metallo-β-lactamases from the B1 and B3 subgroups provide an avenue to combat a major mechanism of antibiotic resistance

Publisher: Elsevier BV

Date: 08-2023

DOI: 10.1016/J.BMCL.2023.129387

Structure, function, and regulation of tartrate-resistant acid phosphatase

Publisher: Elsevier BV

Date: 11-2000

DOI: 10.1016/S8756-3282(00)00368-9

Altering the substrate specificity of methyl parathion hydrolase with directed evolution

Publisher: Elsevier BV

Date: 05-2015

DOI: 10.1016/J.ABB.2015.03.012

Abstract: Many organophosphates (OPs) are used as pesticides in agriculture. They pose a severe health hazard due to their inhibitory effect on acetylcholinesterase. Therefore, detoxification of water and soil contaminated by OPs is important. Metalloenzymes such as methyl parathion hydrolase (MPH) from Pseudomonas sp. WBC-3 hold great promise as bioremediators as they are able to hydrolyze a wide range of OPs. MPH is highly efficient towards methyl parathion (1 × 10(6) s(-1) M(-1)), but its activity towards other OPs is more modest. Thus, site saturation mutagenesis (SSM) and DNA shuffling were performed to find mutants with improved activities on ethyl paraxon (6.1 × 10(3) s(-1) M(-1)). SSM was performed on nine residues lining the active site. Several mutants with modest activity enhancement towards ethyl paraoxon were isolated and used as templates for DNA shuffling. Ultimately, 14 multiple-site mutants with enhanced activity were isolated. One mutant, R2F3, exhibited a nearly 100-fold increase in the kcat/Km value for ethyl paraoxon (5.9 × 10(5) s(-1) M(-1)). These studies highlight the 'plasticity' of the MPH active site that facilitates the fine-tuning of its active site towards specific substrates with only minor changes required. MPH is thus an ideal candidate for the development of an enzyme-based bioremediation system.

Synthesis, Magnetic Properties, and Phosphoesterase Activity of Dinuclear Cobalt(II) Complexes

Publisher: American Chemical Society (ACS)

Date: 02-2013

DOI: 10.1021/IC302418X

Abstract: A series of dinuclear cobalt(II) complexes has been prepared and characterized to generate functional and spectroscopic models for cobalt(II) substituted phosphoesterase enzymes such as the potential bioremediator GpdQ. Reaction of ligands based on 2,2'-(((2-hydroxy-5-methyl-1,3-phenylene)bis(methylene))bis((pyridin-2-ylmethyl)azanediyl)))diethanol (L1) and 2,6-bis(((2-methoxyethyl)(pyridin-2-ylmethyl)amino)methyl)-4-methylphenol (L2) with cobalt(II) salts afforded [Co(2)(CO(2)EtH(2)L1)(CH(3)COO)(2)](PF(6)), [Co(2)(CO(2)EtL2)(CH(3)COO)(2)](PF(6)), [Co(2)(CH(3)L2)(CH(3)COO)(2)](PF(6)), [Co(2)(BrL2)(CH(3)COO)(2)](PF(6)), and [Co(2)(NO(2)L2)(CH(3)COO)(2)](PF(6)). Complexes of the L2 ligands contain a coordinated methyl-ether, whereas the L1 ligand contains a coordinated alcohol. The complexes were characterized using mass spectrometry, microanalysis, X-ray crystallography, UV-vis-NIR diffuse reflectance spectroscopy, IR absorption spectroscopy, solid state magnetic susceptibility measurements, and variable-temperature variable-field magnetic circular dichroism (VTVH MCD) spectroscopy. Susceptibility studies show that [Co(2)(CO(2)EtH(2)L1)(CH(3)COO)(2)](PF(6)), [Co(2)(CO(2)EtL2)(CH(3)COO)(2)](PF(6)), and [Co(2)(CH(3)L2)(CH(3)COO)(2)](PF(6)) are weakly antiferromagnetically coupled, whereas [Co(2)(BrL2)(CH(3)COO)(2)](PF(6)) and [Co(2)(NO(2)L2)(CH(3)COO)(2)](PF(6)) are weakly ferromagnetically coupled. The susceptibility results are confirmed by the VTVH MCD studies. Density functional theory calculations revealed that magnetic exchange coupling occurs mainly through the phenolic oxygen bridge. Implications of geometry and ligand design on the magnetic exchange coupling will be discussed. Functional studies of the complexes with the substrate bis(2,4-dinitrophenyl) phosphate showed them to be active towards hydrolysis of phosphoester substrates.

Enhancement of antibiotic-activity through complexation with metal ions - Combined ITC, NMR, enzymatic and biological studies

Publisher: Elsevier BV

Date: 02-2017

DOI: 10.1016/J.JINORGBIO.2016.11.028

Abstract: Alternative solutions need to be developed to overcome the growing problem of multi-drug resistant bacteria. This study explored the possibility of creating complexes of antibiotics with metal ions, thereby increasing their activity. Analytical techniques such as isothermal titration calorimetry and nuclear magnetic resonance were used to examine the structure and interactions between Cu(II), Ag(I) or Zn(II) and β-lactam antibiotics. The metal-β-lactam complexes were also tested for antimicrobial activity, by micro-broth dilution and disk diffusion methods, showing a synergistic increase in the activity of the drugs, and enzymatic inhibition assays confirming inhibition of β-lactamases responsible for resistance. The metal-antibiotic complex concept was proven to be successful with the activity of the drugs enhanced against β-lactamase-producing bacteria. The highest synergistic effects were observed for complexes formed with Ag(I).

3-Mercapto-1,2,4-triazoles and N-acylated thiosemicarbazides as metallo-β-lactamase inhibitors

Publisher: Elsevier BV

Date: 2012

DOI: 10.1016/J.BMCL.2011.10.116

Abstract: The production of β-lactamases is an effective strategy by which pathogenic bacteria can develop resistance against β-lactam antibiotics. While inhibitors of serine-β-lactamases are widely used in combination therapy with β-lactam antibiotics, there are no clinically available inhibitors of metallo-β-lactamases (MBLs), and so there is a need for the development of such inhibitors. This work describes the optimisation of a lead inhibitor previously identified by fragment screening of a compound library. We also report that thiosemicarbazide intermediates in the syntheses of these compounds are also moderately potent inhibitors of the IMP-1 MBL from Pseudomonas aeruginosa. The interactions of these inhibitors with the active site of IMP-1 were examined using in silico methods.

Reaction mechanism of the metallohydrolase CpsB from Streptococcus pneumoniae, a promising target for novel antimicrobial agents.

Publisher: Royal Society of Chemistry (RSC)

Date: 2017

DOI: 10.1039/C7DT01350G

Abstract: CpsB is a metal ion-dependent hydrolase involved in the biosynthesis of capsular polysaccharides in bacterial organisms. The enzyme has been proposed as a promising target for novel chemotherapeutics to combat antibiotic resistance. The crystal structure of CpsB indicated the presence of as many as three closely spaced metal ions, modelled as Mn

Reactivity of M^II Metal-Substituted Derivatives of Pig Purple Acid Phosphatase (Uteroferrin) with Phosphate

Publisher: American Chemical Society (ACS)

Date: 10-10-2002

DOI: 10.1021/IC020037F

Abstract: The Fe(II) of the binuclear Fe(II)Fe(III) active site of pig purple acid phosphatase (uteroferrin) has been replaced in turn by five M(II) ions (Mn(II), Co(II), Ni(II), Cu(II), and Zn(II)). An uptake of 1 equiv of M(II) is observed in all cases except that of Cu(II), when a second more loosely bound Cu(II) is removed by treatment with edta. The products have been characterized by different analytical procedures and by UV-vis spectrophotometry. At 25 degrees C, I = 0.100 M (NaCl), the nonenzymatic reactions with H(2)PO(4)(-) give the mu-phosphato product, and formation constants K/M(-1) show an 8-fold spread at pH 4.9 of 740 (Mn), 165 (Fe), 190 (Co), 90 (Ni), 800 (Cu), 380 (Zn). The variations in K correlate well with stability constants for the complexing of H(2)PO(4)(-) and (CH(3)O)HPO(3)(-) with M(II) hexaaqua ions. At pH 4.9 with [H(2)PO(4)(-)] > or = 3.5 mM rate constants k(obs) decrease, and an inhibition process in which a second [H(2)PO(4)(-)] coordinates to the dinuclear center is proposed. The mechanism considered accounts for most but not all of the features displayed. Thus K(1) values for the coordination of phosphate to M(II) are in the range10-60 M(-1), whereas K(2) values for the bridging of the phosphate to Fe(III) are in the narrower range 7.8-12.4. From the fits described K(i) approximately 10(3) M(-1) for the inhibition step, which is independent of the identity of M(II). Values of k(obs) decrease with increasing pH, giving pK(a) values which are close to 3.8 and independent of M(II) (Fe(II), Zn(II), Mn(II)). The acid dissociation process is assigned to Fe(III)-OH(2) to Fe(III)-OH(-), where OH(-) is less readily displaced by phosphate.

Metallo-β-Lactamases: A Major Threat to Human Health

Publisher: Scientific Research Publishing, Inc.

Date: 2014

DOI: 10.4236/AJMB.2014.43011

Expansin assisted bio-affinity immobilization of endoxylanase from Bacillus subtilis onto corncob residue: Characterization and efficient production of xylooligosaccharides

Publisher: Elsevier BV

Date: 06-2019

DOI: 10.1016/J.FOODCHEM.2019.01.004

Abstract: A one-step method to immobilize xylanase onto cellulosic material by fusion of expansin from Bacillus subtilis to xylanase LC9 without the requirement of prior purification of enzyme has been developed. Fusion enzyme EXLX-R2-XYN was specifically adsorbed onto corncob residue with high loading capacity due to bio-affinity adsorption of expansin onto cellulose. The immobilization yield was close to 100%, with a recovered activity of 82.4%. The immobilized EXLX-R2-XYN retained 45.3% of its activity after incubation at 70 °C for 3 h, whereas only 16.3% of the activity was left in free form under the same conditions. The conversion yield of XOS by using immobilized EXLX-R2-XYN reached up to 515 mg/g xylan from 2% corncob extracted xylan, which was higher than that of the free enzyme. The hydrolysis products were mainly xylobiose (57.5%) and xylotriose (38.4%), without undesirable xylose production. After five cycles of hydrolysis, more than 70% of conversion was obtained.

Synthesis, Magnetic Properties, and Catalytic Properties of a Nickel(II)-Dependent Biomimetic of Metallohydrolases

Publisher: Frontiers Media SA

Date: 25-09-2018

DOI: 10.3389/FCHEM.2018.00441

Spectroscopic Characterization of Soybean Lipoxygenase-1 Mutants:  the Role of Second Coordination Sphere Residues in the Regulation of Enzyme Activity

Publisher: American Chemical Society (ACS)

Date: 30-05-2003

DOI: 10.1021/BI027380G

Abstract: Lipoxygenases are non-heme iron enzymes, which catalyze the stereo- and regiospecific hydroperoxidation of unsaturated fatty acids. Spectroscopic studies on soybean lipoxygenase have shown that the ferrous form of the enzyme is a mixture of five- and six-coordinate species (40 and 60%, respectively). Addition of substrate leads to a purely six-coordinate form. A series of mutations in the second coordination sphere (Q697E, Q697N, Q495A, and Q495E) were generated, and the structures of the mutants were solved by crystallography [Tomchick et al. (2001) Biochemistry 40, 7509-7517]. While this study clearly showed the contribution of H-bond interactions between the first and the second coordination spheres in catalysis, no correlation with the coordination environment of the Fe(II) was observed. A recent study using density-functional theory [Lehnert and Solomon (2002) J. Biol. Inorg. Chem. 8, 294-305] indicated that coordination flexibility, involving the Asn694 ligand, is regulated via H-bond interactions. In this paper, we investigate the solution structures of the second coordination sphere mutants using CD and MCD spectroscopy since these techniques are more sensitive indicators of the first coordination sphere ligation of Fe(II) systems. Our data demonstrate that the iron coordination environment directly relates to activity, with the mutations that have the ability to form a five-coordinate/six-coordinate mixture being more active. We propose that the H-bond between the weak Asn694 ligand and the Gln697 plays a key role in the modulation of the coordination flexibility of Asn694, and thus, is crucial for the regulation of enzyme reactivity.

Synthesis and evaluation of novel purple acid phosphatase inhibitors

Publisher: Royal Society of Chemistry (RSC)

Date: 2019

DOI: 10.1039/C8MD00491A

Abstract: Inhibitors of the binuclear metallohydrolase purple acid phosphatase, with potential application for the treatment of osteoporosis.

Mutation of outer-shell residues modulates metal ion co-ordination strength in a metalloenzyme

Publisher: Portland Press Ltd.

Date: 28-06-2010

DOI: 10.1042/BJ20100233

Abstract: The metal ion co-ordination sites of many metalloproteins have been characterized by a variety of spectroscopic techniques and small-molecule model systems, revealing many important insights into the structural determinants of metal ion co-ordination. However, our understanding of this fundamentally and practically important phenomenon remains frustratingly simplistic in many proteins it is essentially impossible to predict metal ion specificity and the effects of remote ‘outer-shell’ residues on metal ion co-ordination strength are also poorly defined. This is exemplified by our inability to explain why metalloenzymes with identical metal ion co-ordination spheres, such as the closely related orthologues of bacterial PTE (phosphotriesterase) from Agrobacterium radiobacter and Pseudomonas diminuta, display different metal ion specificity and co-ordination strength. In the present study, we present a series of PTE variants that all possess identical metal ion co-ordination spheres, yet display large differences in their metal ion co-ordination strength. Using measurement of the rates of metal ion dissociation from the active site alongside analysis of structural data obtained through X-ray crystallography, we show that ‘outer-shell’ residues provide essential support for the metal ion ligands, in effect buttressing them in their optimal orientation. Remote mutations appear to modulate metal ion interactions by increasing or decreasing the stabilizing effects of these networks. The present study therefore provides a description of how the greater protein fold can be modified to ‘tune’ the strength of metal ion co-ordination and metal ion specificity, as well as reinforcing the concept of proteins as ensembles of conformational states with unique structures and biochemical properties.

Synthesis and Kinetic Testing of Tetrahydropyrimidine‐2‐thione and Pyrrole Derivatives as Inhibitors of the Metallo‐β‐lactamase from Klebsiella pneumonia and Pseudomonas aeruginosa

Publisher: Wiley

Date: 23-07-2012

DOI: 10.1111/J.1747-0285.2012.01440.X

Abstract: Metallo-β-lactamases (MBLs), produced by an increasing number of bacterial pathogens, facilitate the hydrolysis of many commonly used β-lactam antibiotics. There are no clinically useful antagonists against MBLs. Two sets of tetrahydropyrimidine-2-thione and pyrrole derivatives were synthesized and assayed for their inhibitory effects on the catalytic activity of the IMP-1 MBL from Pseudomonas aeruginosa and Klebsiella pneumoniae. Nine compounds tested (1a, 3b, 5c, 6b, 7a, 8a, 11c, 13a, and 16a) showed micromolar inhibition constants (K(i) values range from ∼20-80 μM). Compounds 1c, 2b, and 15a showed only weak inhibition. In silico docking was employed to investigate the binding mode of each enantiomer of the strongest inhibitor, 5c (K(i) = 19 ± 9 μM), as well as 7a (K(i) =21 ± 10 μM), the strongest inhibitor of the pyrrole series, in the active site of IMP-1.

Diesterase Activity and Substrate Binding in Purple Acid Phosphatases

Publisher: American Chemical Society (ACS)

Date: 17-07-2007

DOI: 10.1021/JA072647Q

Elucidating the Roles of Distinct Chemical Factors in the Hydrolytic Activities of Hetero- and Homonuclear Synthetic Analogues of Binuclear Metalloenzymes

Publisher: American Chemical Society (ACS)

Date: 16-02-2023

DOI: 10.1021/ACSCATAL.2C05758

Phosphate-bound structure of an organophosphate-degrading enzyme from Agrobacterium radiobacter

Publisher: Elsevier BV

Date: 2012

DOI: 10.1016/J.JINORGBIO.2011.09.015

Abstract: OpdA is a binuclear metalloenzyme that can hydrolyze organophosphate pesticides and nerve agents. In this study the crystal structure of the complex between OpdA and phosphate has been determined to 2.20 Å resolution. The structure shows the phosphate bound in a tripodal mode to the metal ions whereby two of the oxygen atoms of PO(4) are terminally bound to each metal ion and a third oxygen bridges the two metal ions, thus displacing the μOH in the active site. In silico modelling demonstrates that the phosphate moiety of a reaction product, e.g. diethyl phosphate, may bind in the same orientation, positioning the diethyl groups neatly into the substrate binding pocket close to the metal center. Thus, similar to the binuclear metallohydrolases urease and purple acid phosphatase the tripodal arrangement of PO(4) is interpreted in terms of a role of the μOH as a reaction nucleophile.

The role of Zn-OR and Zn-OH nucleophiles and the influence of para-substituents in the reactions of binuclear phosphatase mimetics

Publisher: Royal Society of Chemistry (RSC)

Date: 2012

DOI: 10.1039/C1DT11187F

Abstract: Analogues of the ligand 2,2'-(2-hydroxy-5-methyl-1,3-phenylene)bis(methylene)bis((pyridin-2-ylmethyl)azanediyl)diethanol (CH(3)H(3)L1) are described. Complexation of these analogues, 2,6-bis(((2-methoxyethyl)(pyridin-2-ylmethyl)amino)methyl)-4-methylphenol (CH(3)HL2), 4-bromo-2,6-bis(((2-methoxyethyl)(pyridin-2-ylmethyl)amino)methyl)phenol (BrHL2), 2,6-bis(((2-methoxyethyl)(pyridin-2-ylmethyl)amino)methyl)-4-nitrophenol (NO(2)HL2) and 4-methyl-2,6-bis(((2-phenoxyethyl)(pyridin-2-ylmethyl)amino)methyl)phenol (CH(3)HL3) with zinc(II) acetate afforded [Zn(2)(CH(3)L2)(CH(3)COO)(2)](PF(6)), [Zn(2)(NO(2)L2)(CH(3)COO)(2)](PF(6)), [Zn(2)(BrL2)(CH(3)COO)(2)](PF(6)) and [Zn(2)(CH(3)L3)(CH(3)COO)(2)](PF(6)), in addition to [Zn(4)(CH(3)L2)(2)(NO(2)C(6)H(5)OPO(3))(2)(H(2)O)(2)](PF(6))(2) and [Zn(4)(BrL2)(2)(PO(3)F)(2)(H(2)O)(2)](PF(6))(2). The complexes were characterized using (1)H and (13)C NMR spectroscopy, mass spectrometry, microanalysis, and X-ray crystallography. The complexes contain either a coordinated methyl- (L2 ligands) or phenyl- (L3 ligand) ether, replacing the potentially nucleophilic coordinated alcohol in the previously reported complex [Zn(2)(CH(3)HL1)(CH(3)COO)(H(2)O)](PF(6)). Functional studies of the zinc complexes with the substrate bis(2,4-dinitrophenyl) phosphate (BDNPP) showed them to be competent catalysts with, for ex le, [Zn(2)(CH(3)L2)](+), k(cat) = 5.70 ± 0.04 × 10(-3) s(-1) (K(m) = 20.8 ± 5.0 mM) and [Zn(2)(CH(3)L3)](+), k(cat) = 3.60 ± 0.04 × 10(-3) s(-1) (K(m) = 18.9 ± 3.5 mM). Catalytically relevant pK(a)s of 6.7 and 7.7 were observed for the zinc(II) complexes of CH(3)L2(-) and CH(3)L3(-), respectively. Electron donating para-substituents enhance the rate of hydrolysis of BDNPP such that k(cat)p-CH(3) > p-Br > p-NO(2). Use of a solvent mixture containing H(2)O(18)/H(2)O(16) in the reaction with BDNPP showed that for [Zn(2)(CH(3)L2)(CH(3)COO)(2)](PF(6)) and [Zn(2)(NO(2)L2)(CH(3)COO)(2)](PF(6)), as well as [Zn(2)(CH(3)HL1)(CH(3)COO)(H(2)O)](PF(6)), the (18)O label was incorporated in the product of the hydrolysis suggesting that the nucleophile involved in the hydrolysis reaction was a Zn-OH moiety. The results are discussed with respect to the potential nucleophilic species (coordinated deprotonated alcohol versus coordinated hydroxide).

Characterization of a highly efficient antibiotic-degrading metallo-beta-lactamase obtained from an uncultured member of a permafrost community

Publisher: Oxford University Press (OUP)

Date: 2017

DOI: 10.1039/C7MT00195A

Abstract: Microorganisms in the permafrost contain a potent mechanism to inactivate antibiotics.

Inhibition of purple acid phosphatase with α-alkoxynaphthylmethylphosphonic acids

Publisher: Elsevier BV

Date: 2009

DOI: 10.1016/J.BMCL.2008.10.125

Abstract: Purple acid phosphatases (PAPs) are binuclear hydrolases that catalyse the hydrolysis of a range of phosphorylated substrates. Human PAP is a major histochemical marker for the diagnosis of osteoporosis. In patients suffering from this disorder, PAP activity contributes to increased bone resorption and, therefore, human PAP is a key target for the development of anti-osteoporotic drugs. This manuscript describes the design and synthesis of derivatives of 1-naphthylmethylphosphonic acids as inhibitors of PAP. The K(i) values of these compounds are as low as 4 microM, the lowest reported to date for a PAP inhibitor.

A new heterobinuclear FeIIICuII complex with a single terminal FeIII–O(phenolate) bond. Relevance to purple acid phosphatases and nucleases

Publisher: Springer Science and Business Media LLC

Date: 21-04-2005

DOI: 10.1007/S00775-005-0635-7

Abstract: A novel heterobinuclear mixed valence complex [Fe(III)Cu(II)(BPBPMP)(OAc)(2)]ClO(4), 1, with the unsymmetrical N(5)O(2) donor ligand 2-bis[{(2-pyridylmethyl)aminomethyl}-6-{(2-hydroxybenzyl)(2-pyridylmethyl)}aminomethyl]-4-methylphenol (H(2)BPBPMP) has been synthesized and characterized. A combination of data from mass spectrometry, potentiometric titrations, X-ray absorption and electron paramagnetic resonance spectroscopy, as well as kinetics measurements indicates that in ethanol/water solutions an [Fe(III)-(mu)OH-Cu(II)OH(2)](+) species is generated which is the likely catalyst for 2,4-bis(dinitrophenyl)phosphate and DNA hydrolysis. Insofar as the data are consistent with the presence of an Fe(III)-bound hydroxide acting as a nucleophile during catalysis, 1 presents a suitable mimic for the hydrolytic enzyme purple acid phosphatase. Notably, 1 is significantly more reactive than its isostructural homologues with different metal composition (Fe(III)M(II), where M(II) is Zn(II), Mn(II), Ni(II), or Fe(II)). Of particular interest is the observation that cleavage of double-stranded plasmid DNA occurs even at very low concentrations of 1 (2.5 microM), under physiological conditions (optimum pH of 7.0), with a rate enhancement of 2.7 x 10(7) over the uncatalyzed reaction. Thus, 1 is one of the most effective model complexes to date, mimicking the function of nucleases.

Copper ions and coordination complexes as novel carbapenem adjuvants

Publisher: American Society for Microbiology

Date: 02-2018

DOI: 10.1128/AAC.02280-17

Abstract: Carbapenem-resistant Enterobacteriaceae are urgent threats to global human health. These organisms produce β-lactamases with carbapenemase activity, such as the metallo-β-lactamase NDM-1, which is notable due to its association with mobile genetic elements and the lack of a clinically useful inhibitor. Here we examined the ability of copper to inhibit the activity of NDM-1 and explored the potential of a copper coordination complex as a mechanism to efficiently deliver copper as an adjuvant in clinical therapeutics. An NDM-positive Escherichia coli isolate, MS6192, was cultured from the urine of a patient with a urinary tract infection. MS6192 was resistant to antibiotics from multiple classes, including erse β-lactams (penicillins, cephalosporins, and carbapenems), aminoglycosides, and fluoroquinolones. In the presence of copper (range, 0 to 2 mM), however, the susceptibility of MS6192 to the carbapenems ertapenem and meropenem increased markedly. In standard checkerboard assays, copper decreased the MICs of ertapenem and meropenem against MS6192 in a dose-dependent manner, suggesting a synergistic mode of action. To examine the inhibitory effect of copper in the absence of other β-lactamases, the bla NDM-1 gene from MS6192 was cloned and expressed in a recombinant E. coli K-12 strain. Analysis of cell extracts prepared from this strain revealed that copper directly inhibited NDM-1 activity, which was confirmed using purified recombinant NDM-1. Finally, delivery of copper at a low concentration of 10 μM by using the FDA-approved coordination complex copper-pyrithione sensitized MS6192 to ertapenem and meropenem in a synergistic manner. Overall, this work demonstrates the potential use of copper coordination complexes as novel carbapenemase adjuvants.

X-Ray Absorption Spectroscopy of Dinuclear Metallohydrolases

Publisher: Elsevier BV

Date: 09-2014

DOI: 10.1016/J.BPJ.2014.07.066

Structural Flexibility Enhances the Reactivity of the Bioremediator Glycerophosphodiesterase by Fine-Tuning Its Mechanism of Hydrolysis

Publisher: American Chemical Society (ACS)

Date: 04-08-2009

DOI: 10.1021/JA903534F

Abstract: The glycerophosphodiesterase from Enterobacter aerogenes (GpdQ) belongs to the family of binuclear metallohydrolases and has attracted recent attention due to its potential in bioremediation. Formation of a catalytically competent binuclear center is promoted by the substrate (Hadler et al. J. Am. Chem. Soc. 2008, 130, 14129). Using the paramagnetic properties of Mn(II), we estimated the K(d) values for the metal ions in the alpha and beta sites to be 29 and 344 microM, respectively, in the absence of a substrate analogue. In its presence, the affinity of the beta site increases substantially (K(d) = 56 microM), while that of the alpha site is not greatly affected (K(d) = 17 microM). Stopped-flow fluorescence measurements identified three distinct phases in the catalytic turnover, associated with the initial binding of substrate to the active site (k(obs1)), the assembly of a catalytically active binuclear center (k(obs2)), and subsequent slower structural rearrangements to optimize catalysis (k(obs3)). These three phases depend on the concentration of substrate ([S]), with k(obs1) and k(obs2) reaching maximum values at high [S] (354 and 38 s(-1), respectively), whereas k(obs3) is reduced as [S] is increased. The k(cat) for the hydrolysis of the substrate bis(para-nitrophenyl) phosphate (approximately 1 s(-1)) gradually increases from the moment of initiating the reaction, reaching a maximum when the structural change associated with k(obs3) is complete. This structural change is mediated via an extensive hydrogen-bond network that connects the coordination sphere with the substrate binding pocket, as demonstrated by mutation of two residues in this network (His81 and His217). The identities of both the substrate and the metal ion also affect interactions within this H-bond network, thus leading to some mechanistic variations. Overall, the mechanism employed by GpdQ is a paradigm of a substrate- and metal-ion-induced fit to optimize catalysis.

Metabolomics of Non-Saccharomyces Yeasts in Fermented Beverages

Publisher: MDPI AG

Date: 20-07-2022

DOI: 10.3390/BEVERAGES8030041

Abstract: Fermented beverages have been consumed for millennia and today support a global industry producing erse products. Saccharomyces yeasts currently dominate the fermented beverage industry, but consumer demands for alternative products with a variety of sensory profiles and actual or perceived health benefits are driving the ersification and use of non-Saccharomyces yeasts. The ersity of flavours, aromas, and other sensory characteristics that can be obtained by using non-Saccharomyces yeasts in fermentation is, in large part, due to the erse secondary metabolites they produce compared to conventional Saccharomyces yeast. Here, we review the use of metabolomic analyses of non-Saccharomyces yeasts to explore their impact on the sensory characteristics of fermented beverages. We highlight several key species currently used in the industry, including Brettanomyces, Torulaspora, Lachancea, and Saccharomycodes, and emphasize the future potential for the use of non-Saccharomyces yeasts in the production of erse fermented beverages.

Engineering highly functional thermostable proteins using ancestral sequence reconstruction

Publisher: Springer Science and Business Media LLC

Date: 22-10-2018

DOI: 10.1038/S41929-018-0159-5

Identification and molecular modeling of a novel, plant-like, human purple acid phosphatase

Publisher: Elsevier BV

Date: 08-2006

DOI: 10.1016/J.GENE.2006.02.031

Abstract: Purple acid phosphatases are a family of binuclear metallohydrolases that have been identified in plants, animals and fungi. Only one isoform of approximately 35 kDa has been isolated from animals, where it is associated with bone resorption and microbial killing through its phosphatase activity, and hydroxyl radical production, respectively. Using the sensitive PSI-BLAST search method, sequences representing new purple acid phosphatase-like proteins have been identified in mammals, insects and nematodes. These new putative isoforms are closely related to the approximately 55 kDa purple acid phosphatase characterized from plants. Secondary structure prediction of the new human isoform further confirms its similarity to a purple acid phosphatase from the red kidney bean. A structural model for the human enzyme was constructed based on the red kidney bean purple acid phosphatase structure. This model shows that the catalytic centre observed in other purple acid phosphatases is also present in this new isoform. These observations suggest that the sequences identified in this study represent a novel subfamily of plant-like purple acid phosphatases in animals and humans.

Broad spectrum antibiotic-degrading metallo-β-lactamases are phylogenetically diverse and widespread in the environment

Publisher: Cold Spring Harbor Laboratory

Date: 16-08-2019

DOI: 10.1101/737403

Abstract: Antibiotic resistance has emerged as a major global health threat. The Zn 2+ -dependent metallo-β-lactamases (MBLs) are of particular concern as they act on the most widely prescribed class of antibiotics, the β-lactams, and are largely unaffected by commonly used β-lactamase antagonists such as clavulanic acid. MBLs are sub ided into three groups (B1 to B3) despite low overall sequence similarity, their catalytic centers are conserved with two closely spaced Zn 2+ binding sites (α and β site). We recovered almost 1500 B3 MBLs from ,000 public microbial genomes representing a wide range of habitats including pristine sites not impacted by human activity. Although homologs were predominantly identified in members of the bacterial phylum Proteobacteria , the recovered B3 MBLs represent a much broader phylogenetic ersity than is currently appreciated based on the study of model pathogens. This includes three active site variants inferred to have arisen from the ancestral B3 enzyme. One of these variants, B3-RQK, is noteworthy for being broadly sensitive to clavulanic acid. Through targeted mutations we demonstrate that the presence of a lysine residue (Lys263) in the β site of the catalytic center of this variant confers sensitivity to this compound. Replacing this lysine with the canonical histidine (His263) found in all other MBLs restored resistance. Crystallographic and computational data reveal that clavulanic acid inhibits B3-RQK MBLs by displacing the Zn 2+ ion in the β site. Therefore, modifying clavulanic acid to effectively interact with His263 may increase the therapeutic range of this widely used antibiotic resistance drug. This study surveys the environmental and phylogenetic ersity of the B3 subgroup of antibiotic-degrading metallo-β-lactamases (MBLs). B3-like MBLs are more widespread in the environment than previously appreciated suggesting multiple unrecognized reservoirs of antibiotic resistance. Three variants of the canonical active site were identified, including B3-RQK, which amongst the B3 MBLs is uniquely inhibited by the antibiotic resistance drug clavulanic acid. We demonstrate that the mode of inhibition involves the displacement of a catalytically essential Zn 2+ ion from the active site. It may thus be possible to modify clavulanic acid so that it can compete with the Zn 2+ ions in other MBLs as well, increasing the therapeutic range of this compound.

Cadmium(II) Complexes: Mimics of Organophosphate Pesticide Degrading Enzymes and Metallo-β-lactamases

Publisher: American Chemical Society (ACS)

Date: 03-07-2012

DOI: 10.1021/IC300687Y

Abstract: Cadmium(II) complexes of ethyl 4-hydroxy-3,5-bis(((2-hydroxyethyl)(pyridin-2-ylmethyl)amino)methyl)benzoate (CO(2)EtH(3)L1) and ethyl 4-hydroxy-3,5-bis(((2-methoxyethyl)(pyridin-2-ylmethyl)amino)methyl)benzoate (CO(2)EtHL2) are described. The two ligands possess an ethyl ester (CO(2)Et-) at the position para to the phenolic -OH CO(2)EtHL2, with methyl ether donors in contrast to potentially nucleophilic alkoxide donors in CO(2)EtH(3)L1, offers a direct comparison of potential ligand-centered nucleophiles. The complex with CO(2)EtH(3)L1 was characterized using (1)H and (13)C NMR spectroscopy, mass spectrometry and microanalysis X-ray crystallography defined a tetranuclear structure [Cd(4)(CO(2)EtH(2)L1)(2)(CH(3)COO)(3.75)Cl(0.25)(H(2)O)(2)](PF(6))(2). Functional studies of the cadmium(II) complexes were undertaken with the substrates bis(2,4-dinitrophenyl)phosphate (BDNPP), and nitrocefin to assess their phosphatase and β-lactamase activities, respectively. The complexes with CO(2)EtH(3)L1 and CO(2)EtHL2 are competent phosphoesterase mimics with K(M) = 9.4 ± 2.1 mM and 10.1 ± 3.4 mM, k(cat) = 9.4 ± 0.2 × 10(-3) s(-1) and 9.7 ± 2.7 × 10(-3) s(-1), respectively. Use of a solvent mixture containing H(2)(18)O/H(2)(16)O in the reaction with BDNPP showed that for the complex with CO(2)EtH(3)L1 the (18)O label was incorporated in the reaction product suggesting that the nucleophile involved is a Cd-OH moiety and not a metal bound alkoxide for CO(2)EtHL2 the presence of the methyl-ether dictates that the active nucleophile must also be a hydroxide. The cadmium(II) complex with CO(2)EtH(3)L1 was furthermore found to be a competent β-lactamase mimic with k(cat) = 1.39 × 10(-2) ± 3 × 10(-3) s(-1), K(M) = 0.11 ± 0.03 mM, and pK(a) = 7.9 ± 0.1. Mass spectral evidence suggested that the active nucleophile in this reaction is the alkoxide lack of β-lactamase activity of the complex with CO(2)EtHL2 supports this assignment. Similar to enzyme-catalyzed reactions, a blue reaction intermediate in the β-lactamase reaction of the CO(2)EtH(3)L1 complex was also identified. It is proposed that the Cd(II) complexes of CO(2)EtH(3)L1 and CO(2)EtHL2 react identically as phosphatases, with a terminal hydroxide as the nucleophile the former exhibits β-lactamase activity with the alkoxide as a nucleophile, while the latter, without a potentially nucleophilic alkoxide, is inactive.

Conformational sampling, catalysis, and evolution of the bacterial phosphotriesterase

Publisher: Proceedings of the National Academy of Sciences

Date: 22-12-2009

DOI: 10.1073/PNAS.0907548106

Abstract: To efficiently catalyze a chemical reaction, enzymes are required to maintain fast rates for formation of the Michaelis complex, the chemical reaction and product release. These distinct demands could be satisfied via fluctuation between different conformational substates (CSs) with unique configurations and catalytic properties. However, there is debate as to how these rapid conformational changes, or dynamics, exactly affect catalysis. As a model system, we have studied bacterial phosphotriesterase (PTE), which catalyzes the hydrolysis of the pesticide paraoxon at rates limited by a physical barrier—either substrate diffusion or conformational change. The mechanism of paraoxon hydrolysis is understood in detail and is based on a single, dominant, enzyme conformation. However, the other aspects of substrate turnover (substrate binding and product release), although possibly rate-limiting, have received relatively little attention. This work identifies “open” and “closed” CSs in PTE and dominant structural transition in the enzyme that links them. The closed state is optimally preorganized for paraoxon hydrolysis, but seems to block access to/from the active site. In contrast, the open CS enables access to the active site but is poorly organized for hydrolysis. Analysis of the structural and kinetic effects of mutations distant from the active site suggests that remote mutations affect the turnover rate by altering the conformational landscape.

A Potentially Polymerizable Heterodinuclear FeIIIZnII Purple Acid Phosphatase Mimic. Synthesis, Characterization, and Phosphate Ester Hydrolysis Studies

Publisher: CSIRO Publishing

Date: 2011

DOI: 10.1071/CH10424

Abstract: An analogue of the purple acid phosphatase biomimetic 2-((bis(pyridin-2-ylmethyl)amino)methyl)-6-(((2-hydroxybenzyl)(pyridin-2-ylmethyl)amino)methyl)-4-methylphenol has been synthesized. The analogue, 2-((bis(pyridin-2-ylmethyl)amino)methyl)-6-(((2-hydroxy-4-(4-vinylbenzyloxy)benzyl)(pyridin-2-ylmethyl)amino)methyl)-4-methylphenol (H2BPBPMPV) possesses a pendant olefin suitable for copolymerization. Complexation with FeIII/ZnII resulted in the complex [FeIIIZnII(BPBPMPV)(CH3COO)2](ClO4), characterized with mass spectrometry, microanalysis, UV/vis, and IR spectrometry. The catalytic activity of the complex toward bis-(2,4-dinitrophenyl) phosphate was determined, resulting in Km of 4.1 ± 0.6 mM, with kcat 3.8 ± 0.2 × 10–3 s–1 and a bell-shaped pH–rate profile with pKa values of 4.31, 5.66, 8.96, the profile exhibiting residual activity above pH 9.5.

Heterologous expression of human transketolase

Publisher: Elsevier BV

Date: 03-1998

DOI: 10.1016/S1357-2725(97)00154-4

Abstract: Transketolase belongs to the family of thiamin diphosphate dependent enzymes. The aim of this study was to establish a bacterial expression system for human transketolase in order to investigate the functional characteristics of mammalian transketolases. The level of recombinant human enzyme expressed in Escherichia coli was modest. Purification of recombinant transketolase and separation from the E. coli enzyme has been greatly simplified by means of a non-cleavable hexa-histidine tag. The highest specific activity was 13.5 U/mg and the K(m) values were 0.27 +/- 0.02 and 0.51 +/- 0.05 mM for the substrates D-xylulose 5-phosphate and D-ribose 5-phosphate, respectively. Binding of cofactors to the apoenzyme showed the expected hysteresis. Without preincubation, the K(m) values for thiamin diphosphate and for Mg2+ were, respectively, 4.1 +/- 0.8 and 2.5 +/- 0.4 microM, but after 1 h of preincubation these values were 85 +/- 16 nM and 0.74 +/- 0.23 microM. The kinetic constants are similar to those of the native enzyme purified from human erythrocytes. Despite the modest expression level the reported system is well suited to a variety of functional studies.

Dihydroxy‐Acid Dehydratases From Pathogenic Bacteria: Emerging Drug Targets to Combat Antibiotic Resistance

Publisher: Wiley

Date: 16-06-2022

DOI: 10.1002/CHEM.202200927

Abstract: There is an urgent global need for the development of novel therapeutics to combat the rise of various antibiotic‐resistant superbugs. Enzymes of the branched‐chain amino acid (BCAA) biosynthesis pathway are an attractive target for novel anti‐microbial drug development. Dihydroxy‐acid dehydratase (DHAD) is the third enzyme in the BCAA biosynthesis pathway. It relies on an Fe−S cluster for catalytic activity and has recently also gained attention as a catalyst in cell‐free enzyme cascades. Two types of Fe−S clusters have been identified in DHADs, i.e. [2Fe−2S] and [4Fe−4S], with the latter being more prone to degradation in the presence of oxygen. Here, we characterise two DHADs from bacterial human pathogens, Staphylococcus aureus and C ylobacter jejuni ( Sa DHAD and Cj DHAD). Purified Sa DHAD and Cj DHAD are virtually inactive, but activity could be reversibly reconstituted in vitro (up to ∼19,000‐fold increase with k cat as high as ∼6.7 s −1 ). Inductively‐coupled plasma‐optical emission spectroscopy (ICP‐OES) measurements are consistent with the presence of [4Fe−4S] clusters in both enzymes. N‐isopropyloxalyl hydroxamate (IpOHA) and aspterric acid are both potent inhibitors for both Sa DHAD ( K i =7.8 and 51.6 μM, respectively) and Cj DHAD ( K i =32.9 and 35.1 μM, respectively). These compounds thus present suitable starting points for the development of novel anti‐microbial chemotherapeutics.

LAM-1 from Lysobacter antibioticus: A potent zinc-dependent activity that inactivates β-lactam antibiotics

Publisher: Elsevier BV

Date: 2022

DOI: 10.1016/J.JINORGBIO.2021.111637

Abstract: Resistance to β-lactam antibiotics, including the "last-resort" carbapenems, has emerged as a major threat to global health. A major resistance mechanism employed by pathogens involves the use of metallo-β-lactamases (MBLs), zinc-dependent enzymes that inactivate most of the β-lactam antibiotics used to treat infections. Variants of MBLs are frequently discovered in clinical environments. However, an increasing number of such enzymes have been identified in microorganisms that are less impacted by human activities. Here, an MBL from Lysobacter antibioticus, isolated from the rhizosphere, has been shown to be highly active toward numerous β-lactam antibiotics. Its activity is higher than that of some of the most effective MBLs linked to hospital-acquired antibiotic resistance and thus poses an interesting system to investigate evolutionary pressures that drive the emergence of such biocatalysts.

Crystal Structures of Staphylococcus aureus Ketol‐Acid Reductoisomerase in Complex with Two Transition State Analogues that Have Biocidal Activity

Publisher: Wiley

Date: 30-11-2017

DOI: 10.1002/CHEM.201704481

Abstract: Ketol-acid reductoisomerase (KARI) is an NAD(P)H and Mg

Guanidine- and purine-functionalized ligands of FeIIIZnII complexes: effects on the hydrolysis of DNA

Publisher: Springer Science and Business Media LLC

Date: 02-07-2019

DOI: 10.1007/S00775-019-01680-3

Abstract: In this paper, the catalytic effects of aminoguanidine and aminopurine groups in the second sphere of a Fe

Probing the role of the divalent metal ion in uteroferrin using metal ion replacement and a comparison to isostructural biomimetics

Publisher: Springer Science and Business Media LLC

Date: 16-10-2007

DOI: 10.1007/S00775-007-0305-Z

Abstract: Purple acid phosphatases (PAPs) are a group of heterovalent binuclear metalloenzymes that catalyze the hydrolysis of phosphomonoesters at acidic to neutral pH. While the metal ions are essential for catalysis, their precise roles are not fully understood. Here, the Fe(III)Ni(II) derivative of pig PAP (uteroferrin) was generated and its properties were compared with those of the native Fe(III)Fe(II) enzyme. The kcat of the Fe(III)Ni(II) derivative (approximately 60 s(-1)) is approximately 20% of that of native uteroferrin, and the Ni(II) uptake is considerably faster than the reconstitution of full enzymatic activity, suggesting a slow conformational change is required to attain optimal reactivity. An analysis of the pH dependence of the catalytic properties of Fe(III)Ni(II) uteroferrin indicates that the mu-hydroxide is the likely nucleophile. Thus, the Ni(II) derivative employs a mechanism similar to that proposed for the Ga(III)Zn(II) derivative of uteroferrin, but different from that of the native enzyme, which uses a terminal Fe(III)-bound nucleophile to initiate catalysis. Binuclear Fe(III)Ni(II) biomimetics with coordination environments similar to the coordination environment of uteroferrin were generated to provide both experimental benchmarks (structural and spectroscopic) and further insight into the catalytic mechanism of hydrolysis. The data are consistent with a reaction mechanism employing an Fe(III)-bound terminal hydroxide as a nucleophile, similar to that proposed for native uteroferrin and various related isostructural biomimetics. Thus, only in the uteroferrin-catalyzed reaction are the precise details of the catalytic mechanism sensitive to the metal ion composition, illustrating the significance of the dynamic ligand environment in the protein active site for the optimization of the catalytic efficiency.

Induction of apoptosis in leukemia cell lines by new copper(II) complexes containing naphthyl groups via interaction with death receptors

Publisher: Elsevier BV

Date: 12-2015

DOI: 10.1016/J.JINORGBIO.2015.09.014

Abstract: The synthesis, physico-chemical characterization and cytotoxicity of four new ligands and their respective copper(II) complexes toward two human leukemia cell lines (THP-1 and U937) are reported (i.e. [(HL1)Cu(μ-Cl)2Cu(HL1)]Cl2·H2O (1), [(H2L2)Cu(μ-Cl)2Cu(H2L2)]Cl2·5H2O (2), [(HL3)Cu(μ-Cl)2Cu(HL3)]Cl2·4H2O (3), [(H2L4)Cu(μ-Cl)2Cu(H2L4)]Cl2·6H2O (4)). Ligands HL1 and HL3 contain two pyridines, amine and alcohol moieties with a naphthyl pendant unit yielding a N3O coordination metal environment. Ligands H2L2 and H2L4 have pyridine, phenol, amine and alcohol groups with a naphthyl pendant unit providing a N2O2 coordination metal environment. These compounds are likely to be dinuclear in the solid state but form mononuclear species in solution. The complexes have an antiproliferative effect against both leukemia cell lines complex (2) exhibits higher activity than cisplatin against U937 (8.20 vs 16.25μmoldm(-3)) and a comparable one against THP-1. These human neoplastic cells are also more susceptible than peripheral blood mononuclear cells (PBMCs) toward the tested compounds. Using C57BL/6 mice an LD50 of 55mgkg(-1) was determined for complex (2), suggesting that this compound is almost four times less toxic than cisplatin (LD50=14.5mgkg(-1)). The mechanism of cell death promoted by ligand H2L2 and by complexes (2) and (4) was investigated by a range of techniques demonstrating that the apoptosis signal triggered at least by complex (2) starts from an extrinsic pathway involving the activation of caspases 4 and 8. This signal is lified by mitochondria with the concomitant release of cytochrome c and the activation of caspase 9.

Properties and functions of the thiamin diphosphate dependent enzyme transketolase

Publisher: Elsevier BV

Date: 12-1998

DOI: 10.1016/S1357-2725(98)00095-8

Abstract: This review highlights recent research on the properties and functions of the enzyme transketolase, which requires thiamin diphosphate and a alent metal ion for its activity. The transketolase-catalysed reaction is part of the pentose phosphate pathway, where transketolase appears to control the non-oxidative branch of this pathway, although the overall flux of labelled substrates remains controversial. Yeast transketolase is one of several thiamin diphosphate dependent enzymes whose three-dimensional structures have been determined. Together with mutational analysis these structural data have led to detailed understanding of thiamin diphosphate catalysed reactions. In the homodimer transketolase the two catalytic sites, where dihydroxyethyl groups are transferred from ketose donors to aldose acceptors, are formed at the interface between the two subunits, where the thiazole and pyrimidine rings of thiamin diphosphate are bound. Transketolase is ubiquitous and more than 30 full-length sequences are known. The encoded protein sequences contain two motifs of high homology one common to all thiamin diphosphate-dependent enzymes and the other a unique transketolase motif. All characterised transketolases have similar kinetic and physical properties, but the mammalian enzymes are more selective in substrate utilisation than the nonmammalian representatives. Since products of the transketolase-catalysed reaction serve as precursors for a number of synthetic compounds this enzyme has been exploited for industrial applications. Putative mutant forms of transketolase, once believed to predispose to disease, have not stood up to scrutiny. However, a modification of transketolase is a marker for Alzheimer's disease, and transketolase activity in erythrocytes is a measure of thiamin nutrition. The cornea contains a particularly high transketolase concentration, consistent with the proposal that pentose phosphate pathway activity has a role in the removal of light-generated radicals.

Structures of fungal and plant acetohydroxyacid synthases

Publisher: Springer Science and Business Media LLC

Date: 08-07-2020

DOI: 10.1038/S41586-020-2514-3

Identification of a non-purple tartrate-resistant acid phosphatase: an evolutionary link to Ser/Thr protein phosphatases?

Publisher: Springer Science and Business Media LLC

Date: 2008

DOI: 10.1186/1756-0500-1-78

Metal Affinity Immobilization of the Processive Endoglucanase EG5C-1 from Bacillus subtilis on a Recyclable pH-Responsive Polymer

Publisher: American Chemical Society (ACS)

Date: 02-06-2021

DOI: 10.1021/ACSSUSCHEMENG.1C02215

Asymmetric zinc(ii) complexes as functional and structural models for phosphoesterases

Publisher: Royal Society of Chemistry (RSC)

Date: 2013

DOI: 10.1039/C3DT50514F

Abstract: We report two asymmetric ligands for the generation of structural and functional dinuclear metal complexes as phosphoesterase mimics. Two zinc(II) complexes, [Zn2(CH3L4)(CH3CO2)2](+) (CH3HL4 = 2-(((2-methoxyethyl)(pyridin-2-ylmethyl)amino)methyl)-4-methyl-6-(((pyridin-2-ylmethyl)amino)methyl)phenol) and [Zn2(CH3L5)(CH3CO2)2](+) (CH3HL5 = 2-(((2-methoxyethyl)(pyridine-2-ylmethyl)amino)methyl)-4-methyl-6-(((pyridin-2-ylmethyl)(4-vinylbenzyl)amino)methyl)phenol) were synthesized and characterized by X-ray crystallography. The structures showed that the ligands enforce a mixed 6,5-coordinate environment in the solid state. (1)H-, (13)C- and (31)P-NMR, mass spectrometry and infrared spectroscopy were used to further characterize the compounds in the solid state and in solution. The zinc(II) complexes hydrolyzed the organophosphate substrate bis-(2,4-dinitrophenol)phosphate (BDNPP), the nucleophile proposed to be a terminal water molecule (pK(a) 7.2). The ligand CH3HL4 was immobilised on Merrifield resin and its zinc(II) complex generated. Infrared spectroscopy, microanalysis and XPS measurements confirmed successful immobilisation, with a catalyst loading of ~1.45 mmol g(-1) resin. The resin bound complex was active towards BDNPP and displayed similar pH dependence to the complex in solution.

Towards a sustainable generation of pseudopterosin-type bioactives

Publisher: Royal Society of Chemistry (RSC)

Date: 2020

DOI: 10.1039/D0GC01697G

Abstract: Pseudopterosins (Ps), marine diterpene glycosides derived from the marine octocoral Antillogorgia elisabethae , have potent anti-inflammatory activity demonstrated in phase II clinical trials.

Land and sea: Addressing the challenges facing inter-regional ecosystems in developing a sustainable bioeconomy

Publisher: Elsevier BV

Date: 11-2021

DOI: 10.1016/J.BIOECO.2021.100017

Electronic and geometric structures of the organophosphate-degrading enzyme from Agrobacterium radiobacter (OpdA)

Publisher: Springer Science and Business Media LLC

Date: 13-04-2011

DOI: 10.1007/S00775-011-0779-6

Abstract: The organophosphate-degrading enzyme from Agrobacterium radiobacter (OpdA) is a highly efficient catalyst for the degradation of pesticides and some nerve agents such as sarin. OpdA requires two metal ions for catalytic activity, and hydrolysis is initiated by a nucleophilic hydroxide that is bound to one of these metal ions. The precise location of this nucleophile has been contentious, with both a terminal and a metal-ion-bridging hydroxide as likely candidates. Here, we employed magnetic circular dichroism to probe the electronic and geometric structures of the Co(II)-reconstituted dinuclear metal center in OpdA. In the resting state the metal ion in the more secluded α site is five-coordinate, whereas the Co(II) in the solvent-exposed β site is predominantly six-coordinate with two terminal water ligands. Addition of the slow substrate diethyl 4-methoxyphenyl phosphate does not affect the α site greatly but lowers the coordination number of the β site to five. A reduction in the exchange coupling constant indicates that substrate binding also triggers a shift of the μ-hydroxide into a pseudoterminal position in the coordination sphere of either the α or the β metal ion. Mechanistic implications of these observations are discussed.

Remdesivir for 5 or 10 Days in Patients with Severe Covid-19

Publisher: Massachusetts Medical Society

Date: 05-11-2020

DOI: 10.1056/NEJMOA2015301

Promiscuous metallo-β-lactamases: MIM-1 and MIM-2 may play an essential role in quorum sensing networks

Publisher: Elsevier BV

Date: 09-2016

DOI: 10.1016/J.JINORGBIO.2015.12.014

Abstract: MIM-1 and MIM-2 are two recently identified metallo-β-lactamases (MBLs) from Novosphingobium pentaromativorans and Simiduia agarivorans, respectively. Since these organisms are non-pathogenic we speculated that the biological role(s) of MIM-1 and MIM-2 may not be related to their MBL activity. Although both sequence comparison and homology modeling indicate that these proteins are homologous to well-known MBLs such as AIM-1, the sequence analysis also indicated that MIM-1 and MIM-2 share similarities with N-acyl homoserine lactonases (AHLases) and glyoxalase II (GLX-II). Steady-state kinetic assays using a series of lactone substrates confirm that MIM-1 and MIM-2 are efficient lactonases, with catalytic efficiencies resembling those of well-known AHLases. Interestingly, unlike their MBL activity the AHLase activity of MIM-1 and MIM-2 is not dependent on the metal ion composition with Zn(II), Co(II), Cu(II), Mn(II) and Ca(II) all being able to reconstitute catalytic activity (with Co(II) being the most efficient). However, these enzymes do not turn over S-lactoylglutathione, a substrate characteristic for GLX-II activity. Since lactonase activity is linked to the process of quorum sensing the bifunctional activity of "non-pathogenic" MBLs such as MIM-1 and MIM-2 may provide insight into one possible evolutionary pathway for the emergence of antibiotic resistance.

Kinetic and Structural Characterization of the First B3 Metallo-β-Lactamase with an Active-Site Glutamic Acid

Publisher: American Society for Microbiology

Date: 17-09-2021

DOI: 10.1128/AAC.00936-21

Abstract: The structural ersity in metallo-β-lactamases (MBLs), especially in the vicinity of the active site, has been a major hurdle in the development of clinically effective inhibitors. Representatives from three variants of the B3 MBL subclass, containing either the canonical HHH/DHH active-site motif (present in the majority of MBLs in this subclass) or the QHH/DHH (B3-Q) or HRH/DQK (B3-RQK) variations were reported previously.

Synthesis, evaluation and structural investigations of potent purple acid phosphatase inhibitors as drug leads for osteoporosis

Publisher: Elsevier BV

Date: 11-2019

DOI: 10.1016/J.EJMECH.2019.111611

Abstract: Purple acid phosphatases (PAPs) are binuclear hydrolases that catalyze the hydrolysis of phosphorylated substrates under acidic to neutral conditions. Elevated serum concentrations of PAP are observed in patients suffering from osteoporosis, identifying this enzyme as a potential target for the development of novel therapeutic agents to treat this disease. α-Alkoxy-substituted naphthylmethylphosphonic acid derivatives have been identified previously as molecules that bind with high affinity to PAPs, and docking studies suggest that longer alkyl chains may increase the binding affinities of such compounds. Here, we synthesized several derivatives and tested their inhibitory effect against pig and red kidney bean PAPs. The most potent inhibitor within this series is the octadecyl derivative, which has a K

Iron, copper, and manganese complexes with in vitro superoxide dismutase and/or catalase activities that keep Saccharomyces cerevisiae cells alive under severe oxidative stress

Publisher: Elsevier BV

Date: 03-2015

DOI: 10.1016/J.FREERADBIOMED.2014.12.005

Abstract: Due to their aerobic lifestyle, eukaryotic organisms have evolved different strategies to overcome oxidative stress. The recruitment of some specific metalloenzymes such as superoxide dismutases (SODs) and catalases (CATs) is of great importance for eliminating harmful reactive oxygen species (hydrogen peroxide and superoxide anion). Using the ligand HPClNOL {1-[bis(pyridin-2-ylmethyl)amino]-3-chloropropan-2-ol}, we have synthesized three coordination compounds containing iron(III), copper(II), and manganese(II) ions, which are also present in the active site of the above-noted metalloenzymes. These compounds were evaluated as SOD and CAT mimetics. The manganese and iron compounds showed both SOD and CAT activities, while copper showed only SOD activity. The copper and manganese in vitro SOD activities are very similar (IC50~0.4 μmol dm(-3)) and about 70-fold higher than those of iron. The manganese compound showed CAT activity higher than that of the iron species. Analyzing their capacity to protect Saccharomyces cerevisiae cells against oxidative stress (H2O2 and the O2(•-) radical), we observed that all compounds act as antioxidants, increasing the resistance of yeast cells mainly due to a reduction of lipid oxidation. Especially for the iron compound, the data indicate complete protection when wild-type cells were exposed to H2O2 or O2(•-) species. Interestingly, these compounds also compensate for both superoxide dismutase and catalase deficiencies their antioxidant activity is metal ion dependent, in the order iron(III)>copper(II)>manganese(II). The protection mechanism employed by the complexes proved to be independent of the activation of transcription factors (such as Yap1, Hsf1, Msn2/Msn4) and protein synthesis. There is no direct relation between the in vitro and the in vivo antioxidant activities.

Engineering indel and substitution variants of diverse and ancient enzymes using Graphical Representation of Ancestral Sequence Predictions (GRASP)

Publisher: Public Library of Science (PLoS)

Date: 24-10-2022

DOI: 10.1371/JOURNAL.PCBI.1010633

Abstract: Ancestral sequence reconstruction is a technique that is gaining widespread use in molecular evolution studies and protein engineering. Accurate reconstruction requires the ability to handle appropriately large numbers of sequences, as well as insertion and deletion (indel) events, but available approaches exhibit limitations. To address these limitations, we developed Graphical Representation of Ancestral Sequence Predictions (GRASP), which efficiently implements maximum likelihood methods to enable the inference of ancestors of families with more than 10,000 members. GRASP implements partial order graphs (POGs) to represent and infer insertion and deletion events across ancestors, enabling the identification of building blocks for protein engineering. To validate the capacity to engineer novel proteins from realistic data, we predicted ancestor sequences across three distinct enzyme families: glucose-methanol-choline (GMC) oxidoreductases, cytochromes P450, and dihydroxy/sugar acid dehydratases (DHAD). All tested ancestors demonstrated enzymatic activity. Our study demonstrates the ability of GRASP (1) to support large data sets over 10,000 sequences and (2) to employ insertions and deletions to identify building blocks for engineering biologically active ancestors, by exploring variation over evolutionary time.

Using directed evolution to improve the solubility of the C‐terminal domain of Escherichia coli aminopeptidase P

Publisher: Wiley

Date: 21-08-2007

DOI: 10.1111/J.1742-4658.2007.06022.X

Abstract: There have been many approaches to solving problems associated with protein solubility. This article describes the application of directed evolution to improving the solubility of the C-terminal metal-binding domain of aminopeptidase P from Escherichia coli. During the course of experiments, the domain boundary and sequence were allowed to vary. It was found that extending the domain boundary resulted in aggregation with little improvement in solubility, whereas two changes to the sequence of the domain resulted in dramatic improvements in solubility. These latter changes occurred in the active site and abolished the ability of the protein to bind metals and hence catalyze its physiological reaction. The evidence presented here has led to the proposal that metals bind to the intact protein after it has folded and that the N-terminal domain is necessary to stabilize the structure of the protein so that it is capable of binding metals. The acid residues responsible for binding metals tend to repel one another - in the absence of the N-terminal domain, the C-terminal domain does not fold properly and forms inclusion bodies. Evolution of the C-terminal domain has removed the destabilizing effects of the metal ligands, but in so doing it has reduced the capacity of the domain to bind metals. In this case, directed evolution has identified active site residues that destabilize the domain structure.

Crystal Structure of the Commercial Herbicide, Amidosulfuron, in Complex with Arabidopsis thaliana Acetohydroxyacid Synthase

Publisher: American Chemical Society (ACS)

Date: 21-03-2023

DOI: 10.1021/ACS.JAFC.2C08528

Spectroscopic and Catalytic Characterization of a Functional Fe^IIIFe^II Biomimetic for the Active Site of Uteroferrin and Protein Cleavage

Publisher: American Chemical Society (ACS)

Date: 30-01-2012

DOI: 10.1021/IC201711P

Abstract: A mixed-valence complex, [Fe(III)Fe(II)L1(μ-OAc)(2)]BF(4)·H(2)O, where the ligand H(2)L1 = 2-{[[3-[((bis(pyridin-2-ylmethyl)amino)methyl)-2-hydroxy-5-methylbenzyl](pyridin-2-ylmethyl)amino]methyl]phenol}, has been studied with a range of techniques, and, where possible, its properties have been compared to those of the corresponding enzyme system purple acid phosphatase. The Fe(III)Fe(II) and Fe(III)(2) oxidized species were studied spectroelectrochemically. The temperature-dependent population of the S = 3/2 spin states of the heterovalent system, observed using magnetic circular dichroism, confirmed that the dinuclear center is weakly antiferromagnetically coupled (H = -2JS(1)·S(2), where J = -5.6 cm(-1)) in a frozen solution. The ligand-to-metal charge-transfer transitions are correlated with density functional theory calculations. The Fe(III)Fe(II) complex is electron paramagnetic resonance (EPR)-silent, except at very low temperatures (<2 K), because of the broadening caused by the exchange coupling and zero-field-splitting parameters being of comparable magnitude and rapid spin-lattice relaxation. However, a phosphate-bound Fe(III)(2) complex showed an EPR spectrum due to population of the S(tot) = 3 state (J= -3.5 cm(-1)). The phosphatase activity of the Fe(III)Fe(II) complex in hydrolysis of bis(2,4-dinitrophenyl)phosphate (k(cat.) = 1.88 × 10(-3) s(-1) K(m) = 4.63 × 10(-3) mol L(-1)) is similar to that of other bimetallic heterovalent complexes with the same ligand. Analysis of the kinetic data supports a mechanism where the initiating nucleophile in the phosphatase reaction is a hydroxide, terminally bound to Fe(III). It is interesting to note that aqueous solutions of [Fe(III)Fe(II)L1(μ-OAc)(2)](+) are also capable of protein cleavage, at mild temperature and pH conditions, thus further expanding the scope of this complex's catalytic promiscuity.

Penicillin inhibitors of purple acid phosphatase

Publisher: Elsevier BV

Date: 04-2012

DOI: 10.1016/J.BMCL.2012.01.123

Abstract: Purple acid phosphatases (PAPs) are binuclear metallohydrolases that have a multitude of biological functions and are found in fungi, bacteria, plants and animals. In mammals, PAP activity is linked with bone resorption and over-expression can lead to bone disorders such as osteoporosis. PAP is therefore an attractive target for the development of drugs to treat this disease. A series of penicillin conjugates, in which 6-aminopenicillanic acid was acylated with aromatic acid chlorides, has been prepared and assayed against pig PAP. The binding mode of most of these conjugates is purely competitive, and some members of this class have potencies comparable to the best PAP inhibitors yet reported. The structurally related penicillin G was shown to be neither an inhibitor nor a substrate for pig PAP. Molecular modelling has been used to examine the binding modes of these compounds in the active site of the enzyme and to rationalise their activities.

Engineering indel and substitution variants of diverse and ancient enzymes using Graphical Representation of Ancestral Sequence Predictions (GRASP)

Publisher: Cold Spring Harbor Laboratory

Date: 31-12-2019

DOI: 10.1101/2019.12.30.891457

Abstract: Ancestral sequence reconstruction is a technique that is gaining widespread use in molecular evolution studies and protein engineering. Accurate reconstruction requires the ability to handle appropriately large numbers of sequences, as well as insertion and deletion (“indel”) events, but available approaches exhibit limitations. To address these limitations, we developed Graphical Representation of Ancestral Sequence Predictions (GRASP), which efficiently implements maximum likelihood methods to enable the inference of ancestors of families with more than 10,000 members. GRASP implements partial order graphs (POGs) to represent and infer insertion and deletion events across ancestors, enabling the identification of building blocks for protein engineering. To validate the capacity to engineer novel proteins from realistic data, we predicted ancestor sequences across three distinct enzyme families: glucose-methanol-choline (GMC) oxidoreductases, cytochromes P450, and dihydroxy/sugar acid dehydratases (DHAD). All tested ancestors demonstrated enzymatic activity. Our study demonstrates the ability of GRASP (1) to support large data sets over 10,000 sequences and (2) to employ insertions and deletions to identify building blocks for engineering biologically active ancestors, by exploring variation over evolutionary time. Massive sequencing projects expose the extent of natural, genetic ersity. Here, we describe a method with capacity to perform ancestor sequence reconstruction from data sets in excess of 10,000 sequences, poised to recover ancestral ersity, including the evolutionary events that determine present-time biological function and structure. We introduce a novel strategy for suggesting “indel variants” that are distinct from, but can be explored alongside, substitution variants for creating ancestral libraries. We demonstrate how indels can be used as building blocks to form “hybrid ancestors” based on this strategy, we synthesise ancestor variants, with varying enzymatic activities, for wide-ranging applications in the biotechnology sector.

Molecular characterization of cyanobacterial short‐chain prenyltransferases and discovery of a novel GGPP phosphatase

Publisher: Wiley

Date: 28-06-2022

DOI: 10.1111/FEBS.16556

Abstract: Cyanobacteria are photosynthetic prokaryotes with strong potential to be used for industrial terpenoid production. However, the key enzymes forming the principal terpenoid building blocks, called short‐chain prenyltransferases (SPTs), are insufficiently characterized. Here, we examined SPTs in the model cyanobacteria Synechococcus elongatus sp. PCC 7942 and Synechocystis sp. PCC 6803. Each species has a single putative SPT (SeCrtE and SyCrtE, respectively). Sequence analysis identified these as type‐II geranylgeranyl pyrophosphate synthases (GGPPSs) with high homology to GGPPSs found in the plastids of green plants and other photosynthetic organisms. In vitro analysis demonstrated that SyCrtE is multifunctional, producing geranylgeranyl pyrophosphate (GGPP C 20 ) primarily but also significant amounts of farnesyl pyrophosphate (FPP, C 15 ) and geranyl pyrophosphate (GPP, C 10 ) whereas SeCrtE appears to produce only GGPP. The crystal structures were solved to 2.02 and 1.37 Å, respectively, and the superposition of the structures against the GGPPS of Synechococcus elongatus sp. PCC 7002 yield a root mean square deviation of 0.8 Å (SeCrtE) and 1.1 Å (SyCrtE). We also discovered that SeCrtE is co‐encoded in an operon with a functional GGPP phosphatase, suggesting metabolic pairing of these two activities and a putative function in tocopherol biosynthesis. This work sheds light on the activity of SPTs and terpenoid synthesis in cyanobacteria. Understanding native prenyl phosphate metabolism is an important step in developing approaches to engineering the production of different chain‐length terpenoids in cyanobacteria.

Detailed stratified GWAS analysis for severe COVID-19 in four European populations

Publisher: Oxford University Press (OUP)

Date: 15-07-2022

DOI: 10.1093/HMG/DDAC158

Abstract: Given the highly variable clinical phenotype of Coronavirus disease 2019 (COVID-19), a deeper analysis of the host genetic contribution to severe COVID-19 is important to improve our understanding of underlying disease mechanisms. Here, we describe an extended genome-wide association meta-analysis of a well-characterized cohort of 3255 COVID-19 patients with respiratory failure and 12 488 population controls from Italy, Spain, Norway and Germany/Austria, including stratified analyses based on age, sex and disease severity, as well as targeted analyses of chromosome Y haplotypes, the human leukocyte antigen region and the SARS-CoV-2 peptidome. By inversion imputation, we traced a reported association at 17q21.31 to a ~0.9-Mb inversion polymorphism that creates two highly differentiated haplotypes and characterized the potential effects of the inversion in detail. Our data, together with the 5th release of summary statistics from the COVID-19 Host Genetics Initiative including non-Caucasian in iduals, also identified a new locus at 19q13.33, including NAPSA, a gene which is expressed primarily in alveolar cells responsible for gas exchange in the lung.

Pesticide degradation by immobilised metalloenzymes provides an attractive avenue for bioremediation

Publisher: Elsevier BV

Date: 11-2021

DOI: 10.1016/J.BIOECO.2021.100015

Catalytic Mechanisms of Metallohydrolases Containing Two Metal Ions

Publisher: Elsevier

Date: 2014

DOI: 10.1016/BS.APCSB.2014.07.002

Abstract: At least one-third of enzymes contain metal ions as cofactors necessary for a erse range of catalytic activities. In the case of polymetallic enzymes (i.e., two or more metal ions involved in catalysis), the presence of two (or more) closely spaced metal ions gives an additional advantage in terms of (i) charge delocalisation, (ii) smaller activation barriers, (iii) the ability to bind larger substrates, (iv) enhanced electrostatic activation of substrates, and (v) decreased transition-state energies. Among this group of proteins, enzymes that catalyze the hydrolysis of ester and amide bonds form a very prominent family, the metallohydrolases. These enzymes are involved in a multitude of biological functions, and an increasing number of them gain attention for translational research in medicine and biotechnology. Their functional versatility and catalytic proficiency are largely due to the presence of metal ions in their active sites. In this chapter, we thus discuss and compare the reaction mechanisms of several closely related enzymes with a view to highlighting the functional ersity bestowed upon them by their metal ion cofactors.

Exploring the correlation between the sequence composition of the nucleotide binding G5 loop of the FeoB GTPase domain (NFeoB) and intrinsic rate of GDP release

Publisher: Portland Press Ltd.

Date: 12-2014

DOI: 10.1042/BSR20140152

Abstract: GDP release from GTPases is usually extremely slow and is in general assisted by external factors, such as association with guanine exchange factors or membrane-embedded GPCRs (G protein-coupled receptors), which accelerate the release of GDP by several orders of magnitude. Intrinsic factors can also play a significant role a single amino acid substitution in one of the guanine nucleotide recognition motifs, G5, results in a drastically altered GDP release rate, indicating that the sequence composition of this motif plays an important role in spontaneous GDP release. In the present study, we used the GTPase domain from EcNFeoB (Escherichia coli FeoB) as a model and applied biochemical and structural approaches to evaluate the role of all the in idual residues in the G5 loop. Our study confirms that several of the residues in the G5 motif have an important role in the intrinsic affinity and release of GDP. In particular, a T151A mutant (third residue of the G5 loop) leads to a reduced nucleotide affinity and provokes a drastically accelerated dissociation of GDP.

Polynuclear zinc(II) complexes of thiosemicarbazone: Synthesis, X-ray structure and biological evaluation

Publisher: Elsevier BV

Date: 02-2020

DOI: 10.1016/J.JINORGBIO.2019.110908

Abstract: Two new dimeric Zn(II) ([{ZnL

Towards high-throughput optimization of microbial lipid production: from strain development to process monitoring

Publisher: Royal Society of Chemistry (RSC)

Date: 2020

DOI: 10.1039/D0SE00540A

Abstract: We describe, for the first time, the use of fast neutron irradiation for the rapid metabolic optimization of an oleaginous yeast. The approach established in this study provides a progressive path towards automatable strain development efforts.

Triazolopyrimidine herbicides are potent inhibitors of Aspergillus fumigatus acetohydroxyacid synthase and potential antifungal drug leads

Publisher: Springer Science and Business Media LLC

Date: 26-10-2021

DOI: 10.1038/S41598-021-00349-9

Abstract: Aspergillus fumigatus is a fungal pathogen whose effects can be debilitating and potentially fatal in immunocompromised patients. Current drug treatment options for this infectious disease are limited to just a few choices (e.g. voriconazole and hotericin B) and these themselves have limitations due to potentially adverse side effects. Furthermore, the likelihood of the development of resistance to these current drugs is ever present. Thus, new treatment options are needed for this infection. A new potential antifungal drug target is acetohydroxyacid synthase (AHAS EC 2.2.1.6), the first enzyme in the branched chain amino acid biosynthesis pathway, and a target for many commercial herbicides. In this study, we have expressed, purified and characterised the catalytic subunit of AHAS from A. fumigatus and determined the inhibition constants for several known herbicides. The most potent of these, penoxsulam and metosulam, have K i values of 1.8 ± 0.9 nM and 1.4 ± 0.2 nM, respectively. Molecular modelling shows that these compounds are likely to bind into the herbicide binding pocket in a mode similar to Candida albicans AHAS. We have also shown that these two compounds inhibit A. fumigatus growth at a concentration of 25 µg/mL. Thus, AHAS inhibitors are promising leads for the development of new anti-aspergillosis therapeutics.

8th IAS Conference on HIV Pathogenesis, Treatment & Prevention 19-22 July 2015, Vancouver, Canada

Publisher: Wiley

Date: 07-2015

DOI: 10.7448/IAS.18.5.20479

Investigation of the identity of the nucleophile initiating the hydrolysis of phosphate esters catalyzed by dinuclear mimics of metallohydrolases

Publisher: Elsevier BV

Date: 09-2016

DOI: 10.1016/J.JINORGBIO.2016.02.008

Abstract: di-Zinc(II) complexes of the ligands 2,6-bis((bis(2-methoxyethyl)amino)methyl)-4-methylphenol (HL1), 2,6-bis(bis(hydroxyethyl)aminomethyl)-4-methylphenol (HL2) and 2,6-bis((hydroxyethyl)(methoxyethyl)-aminomethyl)-4-methylphenol (HL3) have been prepared and characterized. The three ligands differ in their donor types, having ether donors (HL1), alkoxido donors (HL2) and both ether and alkoxido donors (HL3). These differences allowed an investigation into the role of the potential nucleophiles in the hydrolysis reaction with the phosphodiester substrate bis(2,4-dinitrophenyl)phosphate (BDNPP). In addition, the di-Mg(II) complex of ligand HL2 was prepared in order to examine the potential for Mg(II) to replace Zn(II) in these biomimetic systems. Kinetically relevant pK

Adaptation of a continuous, calorimetric kinetic assay to study the agmatinase-catalyzed hydrolytic reaction

Publisher: Elsevier BV

Date: 04-2020

DOI: 10.1016/J.AB.2020.113618

Structural elements that modulate the substrate specificity of plant purple acid phosphatases: Avenues for improved phosphorus acquisition in crops

Publisher: Elsevier BV

Date: 05-2020

DOI: 10.1016/J.PLANTSCI.2020.110445

Cadmium(II) complexes of the glycerophosphodiester-degrading enzyme GpdQ and a biomimetic N,O ligand

Publisher: Springer Science and Business Media LLC

Date: 06-06-2008

DOI: 10.1007/S00775-008-0392-5

Abstract: The glycerophosphodiester-degrading enzyme GpdQ from Enterobacter aerogenes is a promising bioremediator owing to its ability to degrade some organophosphate pesticides and diester products originating from the hydrolysis of nerve agents such as VX. Here, the cadmium derivative of GpdQ was prepared by reconstituting the apoenzyme. Catalytic measurements with (Cd(2+))(2)-GpdQ and the phosphodiester substrate bis(4-nitrophenyl)phosphate yield k(cat) = 15 s(-1). The pK(a) of 9.4, determined from the pH dependence of the catalytic activity, implicates a hydroxide ligand as the catalytic nucleophile. Also prepared was the cadmium-containing biomimetic [Cd(2)((HP)(2)B)(OAc)(2)(OH(2))](PF(6)) (where (HP)(2)B is [2,6-bis([(2-pyridylmethyl)(2-hydroxyethyl)amino]methyl)-4-methylphenol]), which mimics the asymmetry of the metal ion coordination in the active site of GpdQ. The phosphoesterase-like activity of [Cd(2)((HP)(2)B)(OAc)(2)(OH(2))](PF(6)) was studied using the substrate bis(2,4-dinitrophenyl)phosphate, yielding a kinetically relevant pK(a) of 8.9, with k(cat) = 0.004 s(-1). In summary, the model is both an adequate structural and a reasonable functional mimic of GpdQ.

Proteomics Reveals Profound Metabolic Changes in the Alcohol Use Disorder Brain

Publisher: American Chemical Society (ACS)

Date: 26-02-2019

DOI: 10.1021/ACSCHEMNEURO.8B00660

Abstract: Changes in brain metabolism are a hallmark of alcohol use disorder (AUD). Determining how AUD changes the brain proteome is critical for understanding the effects of alcohol consumption on biochemical processes in the brain. We used data-independent acquisition mass spectrometry proteomics to study differences in the abundance of proteins associated with AUD in prefrontal lobe and motor cortex from autopsy brain. AUD had a substantial effect on the overall brain proteome exceeding the inherent differences between brain regions. Proteins associated with glycolysis, trafficking, the cytoskeleton, and excitotoxicity were altered in abundance in AUD. We observed extensive changes in the abundance of key metabolic enzymes, consistent with a switch from glucose to acetate utilization in the AUD brain. We propose that metabolic adaptations allowing efficient acetate utilization contribute to ethanol dependence in AUD.

Functional analysis of the Mn2+ requirement in the catalysis of ureohydrolases arginase and agmatinase - a historical perspective

Publisher: Elsevier BV

Date: 2020

DOI: 10.1016/J.JINORGBIO.2019.110812

Abstract: Ureohydrolases form a conserved family of enzymes with a strict requirement for alent metal ions for catalytic activity. They catalyze the hydrolysis of the guanidino group and produce urea. In their active sites six highly conserved amino acid residues form a binding pocket for two catalytically essential metal ions that are needed to activate a water molecule to initiate the hydrolysis of the guanidino group in a nucleophilic attack. Focus in this review is on two members of the ureohydrolase family, the Mn

Ca^II Binding Regulates and Dominates the Reactivity of a Transition‐Metal‐Ion‐Dependent Diesterase from Mycobacterium tuberculosis

Publisher: Wiley

Date: 14-12-2015

DOI: 10.1002/CHEM.201504001

Abstract: The diesterase Rv0805 from Mycobacterium tuberculosis is a dinuclear metallohydrolase that plays an important role in signal transduction by controlling the intracellular levels of cyclic nucleotides. As Rv0805 is essential for mycobacterial growth it is a promising new target for the development of chemotherapeutics to treat tuberculosis. The in vivo metal-ion composition of Rv0805 is subject to debate. Here, we demonstrate that the active site accommodates two alent transition metal ions with binding affinities ranging from approximately 50 nm for Mn(II) to about 600 nm for Zn(II) . In contrast, the enzyme GpdQ from Enterobacter aerogenes, despite having a coordination sphere identical to that of Rv0805, binds only one metal ion in the absence of substrate, thus demonstrating the significance of the outer sphere to modulate metal-ion binding and enzymatic reactivity. Ca(II) also binds tightly to Rv0805 (Kd ≈40 nm), but kinetic, calorimetric, and spectroscopic data indicate that two Ca(II) ions bind at a site different from the dinuclear transition-metal-ion binding site. Ca(II) acts as an activator of the enzymatic activity but is able to promote the hydrolysis of substrates even in the absence of transition-metal ions, thus providing an effective strategy for the regulation of the enzymatic activity.

Molecular Evolutionary Analysis of the Thiamine-Diphosphate-Dependent Enzyme, Transketolase

Publisher: Springer Science and Business Media LLC

Date: 05-1997

DOI: 10.1007/PL00006179

Abstract: Members of the transketolase group of thiamine-diphosphate-dependent enzymes from 17 different organisms including mammals, yeast, bacteria, and plants have been used for phylogenetic reconstruction. Alignment of the amino acid and DNA sequences for 21 transketolase enzymes and one putative transketolase reveals a number of highly conserved regions and invariant residues that are of predicted importance for enzyme activity, based on the crystal structure of yeast transketolase. One particular sequence of 36 residues has some similarities to the nucleotide-binding motif and we designate it as the transketolase motif. We report further evidence that the recP protein from Streptococcus pneumoniae might be a transketolase and we list a number of invariant residues which might be involved in substrate binding. Phylogenies derived from the nucleotide and the amino acid sequences by various methods show a conventional clustering for mammalian, plant, and gram-negative bacterial transketolases. The branching order of the gram-positive bacteria could not be inferred reliably. The formaldehyde transketolase (sometimes known as dihydroxyacetone synthase) of the yeast Hansenula polymorpha appears to be orthologous to the mammalian enzymes but paralogous to the other yeast transketolases. The occurrence of more than one transketolase gene in some organisms is consistent with several gene duplications. The high degree of similarity in functionally important residues and the fact that the same kinetic mechanism is applicable to all characterized transketolase enzymes is consistent with the proposition that they are all derived from one common ancestral gene. Transketolase appears to be an ancient enzyme that has evolved slowly and might serve as a model for a molecular clock, at least within the mammalian clade.

Enhancing the catalytic activity of a GH5 processive endoglucanase from Bacillus subtilis BS-5 by site-directed mutagenesis

Publisher: Elsevier BV

Date: 2021

DOI: 10.1016/J.IJBIOMAC.2020.12.060

Proteomics reveals profound metabolic changes in the alcohol use disorder brain

Publisher: Cold Spring Harbor Laboratory

Date: 20-10-2018

DOI: 10.1101/447912

Abstract: Changes in brain metabolism are a hallmark of Alcohol Use Disorder (AUD). Determining how AUD changes the brain proteome is critical for understanding the effects of alcohol consumption on biochemical processes in the brain. We used data-independent acquisition mass spectrometry proteomics to study differences in the abundance of proteins associated with AUD in pre-frontal lobe and motor cortex from autopsy brain. AUD had a substantial effect on the overall brain proteome exceeding the inherent differences between brain regions. Proteins associated with glycolysis, trafficking, the cytoskeleton, and excitotoxicity were altered in abundance in AUD. We observed extensive changes in the abundance of key metabolic enzymes, consistent with a switch from glucose to acetate utilization in the AUD brain. We propose that metabolic adaptations allowing efficient acetate utilization contribute to ethanol dependence in AUD.

Cover Feature: Structure‐Guided Modulation of the Catalytic Properties of [2Fe−2S]‐Dependent Dehydratases (ChemBioChem 10/2022)

Publisher: Wiley

Date: 06-04-2022

DOI: 10.1002/CBIC.202200187

Potent Inhibitors Targeting Metallo-β-lactamases from the B1, B2 and B3 Subgroups: Promising Broad-spectrum Agents to Combat a Major Mechanism of Antibiotic Resistance

Publisher: MDPI AG

Date: 31-03-2023

DOI: 10.20944/PREPRINTS202303.0539.V1

Abstract: Metallo-β-lactamases (MBLs) are a group of Zn(II)-dependent enzymes that pose a major threat to global health. They are linked to an increasing number of multi-drug resistant bacterial pathogens, but no clinically useful inhibitor is yet available. Since β-lactam antibiotics, which are inactivated by MBLs, constitute ~65% of all antibiotics used to treat infections, the search for clinically relevant MBL inhibitors is urgent. Here, derivatives of a 2-amino-1-benzyl-4,5-diphenyl-1H-pyrrole-3-carbonitrile (1a) were synthesised and their inhibitory effects assessed against representatives of each of the three subgroups of MBLs (B1, B2, B3). Several compounds are potent inhibitors of each MBL tested, making them excellent candidates for the development of broad-spectrum drug leads. In particular, compound 5f is highly potent across the MBL subfamilies, with Ki values in the low µM range. Furthermore, this compound also dis-plays synergy in combination with antibiotics such as penicillin G, cefuroxime or meropenem. This molecule thus represents one of the most promising compounds developed yet to combat MBLs.

Inhibition studies of ketol-acid reductoisomerases from pathogenic microorganisms

Publisher: Elsevier BV

Date: 10-2020

DOI: 10.1016/J.ABB.2020.108516

An Approach to More Accurate Model Systems for Purple Acid Phosphatases (PAPs)

Publisher: American Chemical Society (ACS)

Date: 21-07-2015

DOI: 10.1021/ACS.INORGCHEM.5B00628

Abstract: The active site of mammalian purple acid phosphatases (PAPs) have a dinuclear iron site in two accessible oxidation states (Fe(III)2 and Fe(III)Fe(II)), and the heterovalent is the active form, involved in the regulation of phosphate and phosphorylated metabolite levels in a wide range of organisms. Therefore, two sites with different coordination geometries to stabilize the heterovalent active form and, in addition, with hydrogen bond donors to enable the fixation of the substrate and release of the product, are believed to be required for catalytically competent model systems. Two ligands and their dinuclear iron complexes have been studied in detail. The solid-state structures and properties, studied by X-ray crystallography, magnetism, and Mössbauer spectroscopy, and the solution structural and electronic properties, investigated by mass spectrometry, electronic, nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR), and Mössbauer spectroscopies and electrochemistry, are discussed in detail in order to understand the structures and relative stabilities in solution. In particular, with one of the ligands, a heterovalent Fe(III)Fe(II) species has been produced by chemical oxidation of the Fe(II)2 precursor. The phosphatase reactivities of the complexes, in particular, also of the heterovalent complex, are reported. These studies include pH-dependent as well as substrate concentration dependent studies, leading to pH profiles, catalytic efficiencies and turnover numbers, and indicate that the heterovalent diiron complex discussed here is an accurate PAP model system.

Structural and functional analysis of a FeoB A143S G5 loop mutant explains the accelerated GDP release rate

Publisher: Wiley

Date: 04-2014

DOI: 10.1111/FEBS.12779

Abstract: GTPases (G proteins) hydrolyze the conversion of GTP to GDP and free phosphate, comprising an integral part of prokaryotic and eukaryotic signaling, protein biosynthesis and cell ision, as well as membrane transport processes. The G protein cycle is brought to a halt after GTP hydrolysis, and requires the release of GDP before a new cycle can be initiated. For eukaryotic heterotrimeric Gαβγ proteins, the interaction with a membrane-bound G protein-coupled receptor catalyzes the release of GDP from the Gα subunit. Structural and functional studies have implicated one of the nucleotide binding sequence motifs, the G5 motif, as playing an integral part in this release mechanism. Indeed, a Gαs G5 mutant (A366S) was shown to have an accelerated GDP release rate, mimicking a G protein-coupled receptor catalyzed release state. In the present study, we investigate the role of the equivalent residue in the G5 motif (residue A143) in the prokaryotic membrane protein FeoB from Streptococcus thermophilus, which includes an N-terminal soluble G protein domain. The structure of this domain has previously been determined in the apo and GDP-bound states and in the presence of a transition state analogue, revealing conformational changes in the G5 motif. The A143 residue was mutated to a serine and analyzed with respect to changes in GTPase activity, nucleotide release rate, GDP affinity and structural alterations. We conclude that the identity of the residue at this position in the G5 loop plays a key role in the nucleotide release rate by allowing the correct positioning and hydrogen bonding of the nucleotide base.

Directed evolution combined with rational design increases activity of GpdQ toward a non-physiological substrate and alters the oligomeric structure of the enzyme

Publisher: Oxford University Press (OUP)

Date: 06-10-2011

DOI: 10.1093/PROTEIN/GZR048

Abstract: Directed evolution was used to enhance the activity of the glycerophosphodiesterase enzyme from Enterobacter aerogenes, GpdQ, toward bis(para-nitrophenol) phosphate (BpNPP), a substrate that is frequently used to assay phosphodiesterases. Native GpdQ has a low level of activity toward BpNPP while the evolved enzymes exhibited k(cat) values that were well over 100 times better while improvements in k(cat)/K(m) of around 500 times were observed along with improved activity we observed a change in the oligomeric structure in the evolved enzymes. The native enzyme is a hexamer with tightly associated dimers related by a 3-fold axis. The stability of the dimer was attributed in part to the cap domain that forms a disulfide bond with its 2-fold-related subunit and in part due to the fact that dimerization results in burying 23.6% of the monomer's accessible surface area. The cap domain also forms the top of the active site and contributes an essential part of the interface between 3-fold-related molecules. The evolved proteins quickly lost one of the cysteine residues that formed the disulfide bond and other mutations that might stabilize the cap domain. The likely effect of these mutations was to open up the active site for the new substrate and to favor the formation of dimeric molecules. The breakdown of the oligomeric structure was accompanied by a reduction in the thermal stability of the protein-as monitored by the residual activity of the native and mutant proteins following pre-incubation at elevated temperatures. A discussion on the evolutionary implications of these studies is presented.

Is Cu^II Coordinated to Patellamides inside Prochloron Cells?

Publisher: Wiley

Date: 26-04-2017

DOI: 10.1002/CHEM.201700895

Abstract: Dinuclear Cu

Identification of Purple Acid Phosphatase Inhibitors by Fragment‐Based Screening: Promising New Leads for Osteoporosis Therapeutics

Publisher: Wiley

Date: 03-09-2012

DOI: 10.1111/CBDD.12001

Abstract: Purple acid phosphatases are metalloenzymes found in animals, plants and fungi. They possess a binuclear metal centre to catalyse the hydrolysis of phosphate esters and anhydrides under acidic conditions. In humans, elevated purple acid phosphatases levels in sera are correlated with the progression of osteoporosis and metabolic bone malignancies, making this enzyme a target for the development of new chemotherapeutics to treat bone-related illnesses. To date, little progress has been achieved towards the design of specific and potent inhibitors of this enzyme that have drug-like properties. Here, we have undertaken a fragment-based screening approach using a 500-compound library identifying three inhibitors of purple acid phosphatases with K(i) values in the 30-60 μm range. Ligand efficiency values are 0.39-0.44 kcal/mol per heavy atom. X-ray crystal structures of these compounds in complex with a plant purple acid phosphatases (2.3-2.7 Å resolution) have been determined and show that all bind in the active site within contact of the binuclear centre. For one of these compounds, the phenyl ring is positioned within 3.5 Å of the binuclear centre. Docking simulations indicate that the three compounds fit into the active site of human purple acid phosphatases. These studies open the way to the design of more potent and selective inhibitors of purple acid phosphatases that can be tested as anti-osteoporotic drug leads.

Asymmetric mono- and dinuclear Ga III and Zn II complexes as models for purple acid phosphatases

Publisher: Elsevier BV

Date: 09-2016

DOI: 10.1016/J.JINORGBIO.2015.12.028

Abstract: Derivatives of the known dinucleating ligands HL

Structural and Catalytic Characterization of a Heterovalent Mn(II)Mn(III) Complex That Mimics Purple Acid Phosphatases

Publisher: American Chemical Society (ACS)

Date: 08-10-2009

DOI: 10.1021/IC9005086

Abstract: The binuclear heterovalent manganese model complex [Mn(II)Mn(III)(L1)(OAc)(2)] ClO(4) x H(2)O (H(2)L1 = 2-(((3-((bis(pyridin-2-ylmethyl)amino)methyl)-2-hydroxy-5-methylbenzyl)(pyridin-2-ylmethyl)amino)-methyl)phenol) has been prepared and studied structurally, spectroscopically, and computationally. The magnetic and electronic properties of the complex have been related to its structure. The complex is weakly antiferromagnetically coupled (J approximately -5 cm(-1), H = -2J S(1) x S(2)) and the electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) spectra identify the Jahn-Teller distortion of the Mn(III) center as predominantly a tetragonal compression, with a significant rhombic component. Electronic structure calculations using density functional theory have confirmed the conclusions derived from the experimental investigations. In contrast to isostructural M(II)Fe(III) complexes (M = Fe, Mn, Zn, Ni), the Mn(II)Mn(III) system is bifunctional possessing both catalase and hydrolase activities, and only one catalytically relevant pK(a) (= 8.2) is detected. Mechanistic implications are discussed.

Highly efficient synthetic iron-dependent nucleases activate both intrinsic and extrinsic apoptotic death pathways in leukemia cancer cells

Publisher: Elsevier BV

Date: 11-2013

DOI: 10.1016/J.JINORGBIO.2013.07.019

Abstract: The nuclease activity and the cytotoxicity toward human leukemia cancer cells of iron complexes, [Fe(HPClNOL)Cl2]NO3 (1), [Cl(HPClNOL)Fe(μ-O)Fe(HPClNOL)Cl]Cl2·2H2O (2), and [(SO4)(HPClNOL)Fe(μ-O)Fe(HPClNOL)(SO4)]·6H2O (3) (HPClNOL=1-(bis-pyridin-2-ylmethyl-amino)-3-chloropropan-2-ol), were investigated. Each complex was able to promote plasmid DNA cleavage and change the supercoiled form of the plasmid to circular and linear ones. Kinetic data revealed that (1), (2) and (3) increase the rate of DNA hydrolysis about 278, 192 and 339 million-fold, respectively. The activity of the complexes was inhibited by distamycin, indicating that they interact with the minor groove of the DNA. The cytotoxic activity of the complexes toward U937, HL-60, Jukart and THP-1 leukemia cancer cells was studied employing 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), fluorescence and electronic transmission microscopies, flow cytometry and a cytochrome C release assay. Compound (2) has the highest activity toward cancer cells and is the least toxic for normal ones (i.e. peripheral blood mononuclear cells (PBMCs)). In contrast, compound (1) is the least active toward cancer cells but displays the highest toxicity toward normal cells. Transmission electronic microscopy indicates that cell death shows features typical of apoptotic cells, which was confirmed using the annexin V-FITC/PI (fluorescein isothiocyanate ropidium iodide) assay. Furthermore, our data demonstrate that at an early stage during the treatment with complex (2) mitochondria lose their transmembrane potential, resulting in cytochrome C release. A quantification of caspases 3, 9 (intrinsic apoptosis pathway) and caspase 8 (extrinsic apoptosis pathway) indicated that both the intrinsic (via mitochondria) and extrinsic (via death receptors) pathways are involved in the apoptotic stimuli.

Engineering proton conductivity in melanin using metal doping

Publisher: Royal Society of Chemistry (RSC)

Date: 2020

DOI: 10.1039/D0TB01390K

Abstract: The proton conductivity in the model bioelectronic material melanin, is increased via a unique doping strategy utilising the chelation of the transition metal ion copper II. We also propose a potential mechanism for future such ionic studies.

Crystallization and preliminary X-ray diffraction data for a purple acid phosphatase from sweet potato

Publisher: International Union of Crystallography (IUCr)

Date: 12-1999

DOI: 10.1107/S0907444999012597

Abstract: Purple acid phosphatase from sweet potato is a homodimer of 110 kDa. Two forms of the enzyme have been characterized. One contains an Fe–Zn centre similar to that previously reported for red kidney bean purple acid phosphatase. Another isoform, the subject of this work, is the first confirmed ex le of an Fe–Mn-containing enzyme. Crystals of this protein have been grown from PEG 6000. They have unit-cell parameters a = b = 118.4, c = 287.4 Å and have the symmetry of space group P 6 5 22, with one dimer per asymmetric unit. Diffraction data collected using a conventional X-ray source from a cryocooled crystal extend to 2.90 Å resolution. The three-dimensional structure of the enzyme will provide insight into the coordination of this novel binuclear metal centre.

Inside Back Cover: Metal Ions Play an Essential Catalytic Role in the Mechanism of Ketol–Acid Reductoisomerase (Chem. Eur. J. 22/2016)

Publisher: Wiley

Date: 04-05-2016

DOI: 10.1002/CHEM.201601889

Investigating coordination flexibility of glycerophosphodiesterase (GpdQ) through interactions with mono-, di-, and triphosphoester (NPP, BNPP, GPE, and paraoxon) substrates

Publisher: Royal Society of Chemistry (RSC)

Date: 2019

DOI: 10.1039/C8CP07031H

Abstract: Interactions of the catalytically active binuclear form of glycerophosphodiesterase (GpdQ) with chemically erse substrates, i.e. phosphomono-, phosphodi-, and phosphotriester have been investigated using molecular dynamics (MD) simulations.

Structure and mechanism of potent bifunctional β-lactam- and homoserine lactone-degrading enzymes from marine microorganisms

Publisher: Springer Science and Business Media LLC

Date: 30-07-2020

DOI: 10.1038/S41598-020-68612-Z

Abstract: Genes that confer antibiotic resistance can rapidly be disseminated from one microorganism to another by mobile genetic elements, thus transferring resistance to previously susceptible bacterial strains. The misuse of antibiotics in health care and agriculture has provided a powerful evolutionary pressure to accelerate the spread of resistance genes, including those encoding β-lactamases. These are enzymes that are highly efficient in inactivating most of the commonly used β-lactam antibiotics. However, genes that confer antibiotic resistance are not only associated with pathogenic microorganisms, but are also found in non-pathogenic (i.e. environmental) microorganisms. Two recent ex les are metal-dependent β-lactamases (MBLs) from the marine organisms Novosphingobium pentaromativorans and Simiduia agarivorans. Previous studies have demonstrated that their β-lactamase activity is comparable to those of well-known MBLs from pathogenic sources (e.g. NDM-1, AIM-1) but that they also possess efficient lactonase activity, an activity associated with quorum sensing. Here, we probed the structure and mechanism of these two enzymes using crystallographic, spectroscopic and fast kinetics techniques. Despite highly conserved active sites both enzymes demonstrate significant variations in their reaction mechanisms, highlighting both the extraordinary ability of MBLs to adapt to changing environmental conditions and the rather promiscuous acceptance of erse substrates by these enzymes.

Syntheticin vivocompartmentalisation improves metabolic flux and modulates the product profile of promiscuous enzymes

Publisher: Cold Spring Harbor Laboratory

Date: 25-11-2022

DOI: 10.1101/2022.11.24.517869

Abstract: Enzyme spatial organisation and compartmentalisation are naturally evolved mechanisms for facilitating multi-step biocatalysis. We explored the synthetic in vivo co-encapsulation of two different cargo proteins in yeast using a self-assembling virus-like particle. Co-encapsulation was verified using single particle techniques for both end-to-end fusion of the cargo proteins with the encapsulation anchor at one end, and coexpression of each cargo protein with their in idual anchors. The co-encapsulation of a bifunctional geranyl diphosphate/farnesyl diphosphate synthase and a bifunctional linalool/nerolidol synthase delivered nerolidol titres up to 30 times that of an unorganised ‘free’ enzyme control, a remarkable improvement from a single engineering step. Interestingly, striking differences in the ratio of products (linalool and nerolidol) were observed with each spatial organisation approach. This work presents the largest reported titre fold increases from in vivo enzyme compartmentalisation and suggests that enzyme spatial organisation could be used to modulate the product profile of promiscuous enzymes.

Metallohydrolase biomimetics with catalytic and structural flexibility

Publisher: Royal Society of Chemistry (RSC)

Date: 2016

DOI: 10.1039/C6DT03200A

Abstract: The phosphatase activity of zinc complexes containing six- and seven-dentate ligands was evaluated through kinetic and 31 P NMR studies.

Human tartrate-resistant acid phosphatase becomes an effective ATPase upon proteolytic activation

Publisher: Elsevier BV

Date: 07-2005

DOI: 10.1016/J.ABB.2005.05.013

Abstract: Proteolytic cleavage in an exposed loop of human tartrate-resistant acid phosphatase (TRAcP) with trypsin leads to a significant increase in activity. At each pH value between 3.25 and 8.0 the cleaved enzyme is more active. Substrate specificity is also influenced by proteolysis. Only the cleaved form is able to hydrolyze unactivated substrates efficiently, and at pH >6 cleaved TRAcP acquires a marked preference for ATP. The cleaved enzyme also has altered sensitivity to inhibitors. Interestingly, the magnitude and mode of inhibition by fluoride depends not only on the proteolytic state but also pH. The combined kinetic data imply a role of the loop residue D158 in catalysis in the cleaved enzyme. Notably, at low pH this residue may act as a proton donor for the leaving group. In this respect the mechanism of cleaved TRAcP resembles that of sweet potato purple acid phosphatase.

Discovery, Synthesis and Evaluation of a Ketol-Acid Reductoisomerase Inhibitor.

Publisher: Wiley

Date: 07-2020

DOI: 10.1002/CHEM.202000899

Purple acid phosphatase: A journey into the function and mechanism of a colorful enzyme

Publisher: Elsevier BV

Date: 2013

DOI: 10.1016/J.CCR.2012.03.020