Thomas Peat

Structural and biochemical analyses of a Clostridium perfringens sortase D transpeptidase

Publisher: International Union of Crystallography (IUCr)

Date: 30-06-2015

DOI: 10.1107/S1399004715009219

Abstract: The assembly and anchorage of various pathogenic proteins on the surface of Gram-positive bacteria is mediated by the sortase family of enzymes. These cysteine transpeptidases catalyze a unique sorting signal motif located at the C-terminus of their target substrate and promote the covalent attachment of these proteins onto an amino nucleophile located on another protein or on the bacterial cell wall. Each of the six distinct classes of sortases displays a unique biological role, with sequential activation of multiple sortases often observed in many Gram-positive bacteria to decorate their peptidoglycans. Less is known about the members of the class D family of sortases (SrtD), but they have a suggested role in spore formation in an oxygen-limiting environment. Here, the crystal structure of the SrtD enzyme from Clostridium perfringens was determined at 1.99 Å resolution. Comparative analysis of the C. perfringens SrtD structure reveals the typical eight-stranded β-barrel fold observed in all other known sortases, along with the conserved catalytic triad consisting of cysteine, histidine and arginine residues. Biochemical approaches further reveal the specifics of the SrtD catalytic activity in vitro , with a significant preference for the LPQTGS sorting motif. Additionally, the catalytic activity of SrtD is most efficient at 316 K and can be further improved in the presence of magnesium cations. Since C. perfringens spores are heat-resistant and lead to foodborne illnesses, characterization of the spore-promoting sortase SrtD may lead to the development of new antimicrobial agents.

Crystallization: digging into the past to learn lessons for the future.

Publisher: Springer New York

Date: 22-11-2015

DOI: 10.1007/978-1-4939-2230-7_8

Abstract: Crystals of biological macromolecules have been observed and grown for well over a century. More effort has been put into biological crystallization in the last few decades due to the importance of X-ray crystal structures, the advent of synchrotron radiation sources, improved computational speed, better software, and the availability of recombinant protein. Here we focus on two important areas of crystal growth: firstly, on techniques for stabilizing the protein s le, and secondly, on strategies and approaches for selecting the crystallization cocktails most suitable for different strategies.

Characterization of the novel broad-spectrum kinase inhibitor CTx-0294885 as an affinity reagent for mass spectrometry-based kinome profiling.

Publisher: American Chemical Society (ACS)

Date: 25-06-2013

DOI: 10.1021/PR3008495

Identification and characterization of two families of F420H2-dependent reductases from Mycobacteria that catalyse aflatoxin degradation

Publisher: Wiley

Date: 16-09-2010

DOI: 10.1111/J.1365-2958.2010.07356.X

The evolving story of AtzT, a periplasmic binding protein

Publisher: International Union of Crystallography (IUCr)

Date: 31-10-2019

DOI: 10.1107/S2059798319013883

Abstract: Atrazine is an s -triazine-based herbicide that is used in many countries around the world in many millions of tons per year. A small number of organisms, such as Pseudomonas sp. strain ADP, have evolved to use this modified s -triazine as a food source, and the various genes required to metabolize atrazine can be found on a single plasmid. The atomic structures of seven of the eight proteins involved in the breakdown of atrazine by Pseudomonas sp. strain ADP have been determined by X-ray crystallography, but the structures of the proteins required by the cell to import atrazine for use as an energy source are still lacking. The structure of AtzT, a periplasmic binding protein that may be involved in the transport of a derivative of atrazine, 2-hydroxyatrazine, into the cell for mineralization, has now been determined. The structure was determined by SAD phasing using an ethylmercury phosphate derivative that diffracted X-rays to beyond 1.9 Å resolution. `Native' (guanine-bound) and 2-hydroxyatrazine-bound structures were also determined to high resolution (1.67 and 1.65 Å, respectively), showing that 2-hydroxyatrazine binds in a similar way to the purportedly native ligand. Structural similarities led to the belief that it may be possible to evolve AtzT from a purine-binding protein to a protein that can bind and detect atrazine in the environment.

A new crystal form of human vascular adhesion protein 1

Publisher: International Union of Crystallography (IUCr)

Date: 16-11-2010

DOI: 10.1107/S1744309110041515

An unexpected vestigial protein complex reveals the evolutionary origins of an s-triazine catabolic enzyme

Publisher: Elsevier BV

Date: 05-2018

DOI: 10.1074/JBC.RA118.001996

A structural genomics approach to the study of quorum sensing: Crystal structures of three LuxS orthologs

Publisher: Elsevier BV

Date: 06-2001

DOI: 10.1016/S0969-2126(01)00613-X

Abstract: Quorum sensing is the mechanism by which bacteria control gene expression in response to cell density. Two major quorum-sensing systems have been identified, system 1 and system 2, each with a characteristic signaling molecule (autoinducer-1, or AI-1, in the case of system 1, and AI-2 in system 2). The luxS gene is required for the AI-2 system of quorum sensing. LuxS and AI-2 have been described in both Gram-negative and Gram-positive bacterial species and have been shown to be involved in the expression of virulence genes in several pathogens. The structure of the LuxS protein from three different bacterial species with resolutions ranging from 1.8 A to 2.4 A has been solved using an X-ray crystallographic structural genomics approach. The structure of LuxS reported here is seen to have a new alpha-beta fold. In all structures, an equivalent homodimer is observed. A metal ion identified as zinc was seen bound to a Cys-His-His triad. Methionine was found bound to the protein near the metal and at the dimer interface. These structures provide support for a hypothesis that explains the in vivo action of LuxS. Specifically, acting as a homodimer, the protein binds a methionine analog, S-ribosylhomocysteine (SRH). The zinc atom is in position to cleave the ribose ring in a step along the synthesis pathway of AI-2.

The DINGO dataset: a comprehensive set of data for the SAMPL challenge

Publisher: Springer Science and Business Media LLC

Date: 21-12-2012

DOI: 10.1007/S10822-011-9521-2

Reverse engineering

Publisher: Royal Society of Chemistry (RSC)

Date: 2017

DOI: 10.1039/C7GC02343J

Abstract: The use of ancestral sequence reconstruction to design novel biocatalysts with improved catalytic properties for the production of polyamide precursors.

Lipidic Cubic Phase-Induced Membrane Protein Crystallization: Interplay Between Lipid Molecular Structure, Mesophase Structure and Properties, and Crystallogenesis

Publisher: American Chemical Society (ACS)

Date: 29-08-2017

DOI: 10.1021/ACS.CGD.7B00519

Intramolecular oxidative deselenization of acylselenoureas: a facile synthesis of benzoxazole amides and carbonic anhydrase inhibitors

Publisher: Royal Society of Chemistry (RSC)

Date: 2016

DOI: 10.1039/C6OB02299E

Abstract: A mild, efficient and one pot procedure to access benzoxazoles using easily accessible acylselenoureas as starting materials has been discovered.

Structure-guided selection of puromycin N-acetyltransferase mutants for enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins.

Publisher: Springer Science and Business Media LLC

Date: 04-03-2021

DOI: 10.1038/S41598-021-84551-9

Abstract: Puromycin and the Streptomyces alboniger -derived puromycin N -acetyltransferase (PAC) enzyme form a commonly used system for selecting stably transfected cultured cells. The crystal structure of PAC has been solved using X-ray crystallography, revealing it to be a member of the GCN5-related N -acetyltransferase (GNAT) family of acetyltransferases. Based on structures in complex with acetyl-CoA or the reaction products CoA and acetylated puromycin, four classes of mutations in and around the catalytic site were designed and tested for activity. Single-residue mutations were identified that displayed a range of enzymatic activities, from complete ablation to enhanced activity relative to wild-type (WT) PAC. Cell pools of stably transfected HEK293 cells derived using two PAC mutants with attenuated activity, Y30F and A142D, were found to secrete up to three-fold higher levels of a soluble, recombinant target protein than corresponding pools derived with the WT enzyme. A third mutant, Y171F, appeared to stabilise the intracellular turnover of PAC, resulting in an apparent loss of selection stringency. Our results indicate that the structure-guided manipulation of PAC function can be utilised to enhance selection stringency for the derivation of mammalian cell lines secreting elevated levels of recombinant proteins.

Structural Basis for a New Mechanism of Inhibition of H I V-1 Integrase Identified by Fragment Screening and Structure-Based Design

Publisher: SAGE Publications

Date: 04-2011

DOI: 10.3851/IMP1716

Abstract: HIV-1 integrase is a clinically validated therapeutic target for the treatment of HIV-1 infection, with one approved therapeutic currently on the market. This enzyme represents an attractive target for the development of new inhibitors to HIV-1 that are effective against the current resistance mutations. A fragment-based screening method employing surface plasmon resonance and NMR was initially used to detect interactions between integrase and fragments. The binding sites of the fragments were elucidated by crystallography and the structural information used to design and synthesize improved ligands. The location of binding of fragments to the catalytic core of integrase was found to be in a previously undescribed binding site, adjacent to the mobile loop. Enzyme assays confirmed that formation of enzyme–fragment complexes inhibits the catalytic activity of integrase and the structural data was utilized to further develop these fragments into more potent novel enzyme inhibitors. We have defined a new site in integrase as a valid region for the structure-based design of allosteric integrase inhibitors. Using a structure-based design process we have improved the activity of the initial fragments 45-fold.

Parallel Screening of Low Molecular Weight Fragment Libraries: Do Differences in Methodology Affect Hit Identification?

Publisher: Elsevier BV

Date: 02-2013

DOI: 10.1177/1087057112465979

Abstract: Fragment screening is becoming widely accepted as a technique to identify hit compounds for the development of novel lead compounds. In neighboring laboratories, we have recently, and independently, performed a fragment screening c aign on the HIV-1 integrase core domain (IN) using similar commercially purchased fragment libraries. The two c aigns used different screening methods for the preliminary identification of fragment hits one used saturation transfer difference nuclear magnetic resonance spectroscopy (STD-NMR), and the other used surface plasmon resonance (SPR) spectroscopy. Both initial screens were followed by X-ray crystallography. Using the STD-NMR/X-ray approach, 15 IN/fragment complexes were identified, whereas the SPR/X-ray approach found 6 complexes. In this article, we compare the approaches that were taken by each group and the results obtained, and we look at what factors could potentially influence the final results. We find that despite using different approaches with little overlap of initial hits, both approaches identified binding sites on IN that provided a basis for fragment-based lead discovery and further lead development. Comparison of hits identified in the two studies highlights a key role for both the conditions under which fragment binding is measured and the criteria selected to classify hits.

The nanoscience behind the art of in-meso crystallization of membrane proteins

Publisher: Royal Society of Chemistry (RSC)

Date: 2017

DOI: 10.1039/C6NR07634C

Abstract: The structural changes occurring at the nanoscale level within the lipid bilayer and driving the in-meso formation of large well-diffracting membrane protein crystals have been uniquely characterized for a model membrane protein, intimin. Importantly, the order to order transitions taking place within the bilayer and the lipidic nanostructures required for crystal growth have been shown to be general, occurring for both the cubic and the sponge mesophase crystallization pathways. For the first time, a transient fluid lamellar phase has been observed and unambiguously assigned for both crystallization pathways, present at the earliest stages of protein crystallogenesis but no longer observed once the crystals surpass the size of the average lyotropic liquid crystalline domain. The reported time-resolved structural investigation provides a significantly improved and general understanding of the nanostructural changes taking place within the mesophase during in-meso crystallization which is a fundamental advance in the enabling area of membrane protein structural biology.

A PMMA microfluidic droplet platform for in vitro protein expression using crude E. coli S30 extract

Publisher: Royal Society of Chemistry (RSC)

Date: 2009

DOI: 10.1039/B911581A

Abstract: Droplet based microfluidics are promising new tools for biological and chemical assays. In this paper, a high throughput and high sensitivity microfluidic droplet platform is described for in vitro protein expression using crude Escherichia coli S30 extract. A flow-focusing polymethylmethacrylate (PMMA) microchip was designed and integrated with different functions involving droplet generation, storage, separation and detection. The material used for the chip is superior to the previously tested polydimethylsiloxane (PDMS) due to its mechanical and chemical properties. Droplet formation characteristics such as size and generation rate are investigated systematically. The effect of surfactants Abil EM90 and Span80 in the oil phase on droplet formation and optical detection is also studied. The performance of the system is demonstrated by the high throughput and stable droplet generation and ultralow detection limit. The robustness of the system is also demonstrated by the successful synthesis of a green fluorescent protein (GFP) using E. coli S30 extract as a source of RNA translation reagents.

Implementation of the Australian Hospital Patient Experience Question Set (AHPEQS): a consumer-driven patient survey

Publisher: CSIRO Publishing

Date: 03-08-2021

DOI: 10.1071/AH20265

Abstract: Objective In 2018 the Australian Commission on Quality and Safety in Health Care released a new consumer driven Australian Hospital Patient Experience Question set. The objective was to explore the acceptability, adoption, appropriateness, feasibility, fidelity, penetration, resources and sustainability of implementing the AHPEQS, and to review which questions correlated with overall rating of care. Methods Thirty-six Australian private hospitals participated in the AHPEQS implementation over an 18-month period. Results Responses were analysed for 86 180 patient experience surveys. Positive patient experiences (Question 12) correlated most highly with Question 2 (My in idual needs were met correlation coefficient (CC) 0.45, P 0.001), Question 4 (I felt cared for CC 0.45, P 0.001) and Question 9 (When I was in the hospital, I felt confident in the safety of my treatment and care CC 0.44, P 0.001). Day procedure patients rated their experience higher than overnight patients. Uptake was rapid across the 36 hospitals, with minimal resources and demonstrable acceptability, adoption, appropriateness, feasibility, fidelity, penetration and sustainability. Conclusion Utilising a consumer-driven survey highlighting treatment and care, assisted in enhancing staff engagement and continuous improvement in patient experience in acute, day procedure and rehabilitation hospitals. What is known about the topic? Patient experience in hospital is associated with healthcare quality, safety and outcomes. What does this paper add? From a consumer perspective, feeling cared for, having needs met and confidence in the safety of care, correlated with a positive patient experience. What are the implications for practitioners? Investing staff time and health service resources into a consumer-driven patient experience survey tool, which highlighted treatment and care, assisted in enhancing staff engagement and continuous improvement in patient experience in acute, day procedure and rehabilitation hospitals.

X-Ray structure of the amidase domain of AtzF, the allophanate hydrolase from the cyanuric acid-mineralizing multienzyme complex

Publisher: American Society for Microbiology

Date: 15-01-2015

DOI: 10.1128/AEM.02783-14

Abstract: The activity of the allophanate hydrolase from Pseudomonas sp. strain ADP, AtzF, provides the final hydrolytic step for the mineralization of s -triazines, such as atrazine and cyanuric acid. Indeed, the action of AtzF provides metabolic access to two of the three nitrogens in each triazine ring. The X-ray structure of the N-terminal amidase domain of AtzF reveals that it is highly homologous to allophanate hydrolases involved in a different catabolic process in other organisms (i.e., the mineralization of urea). The smaller C-terminal domain does not appear to have a physiologically relevant catalytic function, as reported for the allophanate hydrolase of Kluyveromyces lactis , when purified enzyme was tested in vitro . However, the C-terminal domain does have a function in coordinating the quaternary structure of AtzF. Interestingly, we also show that AtzF forms a large, ca. 660-kDa, multienzyme complex with AtzD and AtzE that is capable of mineralizing cyanuric acid. The function of this complex may be to channel substrates from one active site to the next, effectively protecting unstable metabolites, such as allophanate, from solvent-mediated decarboxylation to a dead-end metabolic product.

X-ray crystallography of Epacadostat in adduct with Carbonic Anhydrase IX

Publisher: Elsevier BV

Date: 04-2020

DOI: 10.1016/J.BIOORG.2020.103669

Tapping the Protein Data Bank for crystallization information

Publisher: International Union of Crystallography (IUCr)

Date: 19-11-2005

DOI: 10.1107/S0907444905033202

Determination of the structure of the catabolic N-succinylornithine transaminase (AstC) from Escherichia coli

Publisher: Public Library of Science (PLoS)

Date: 06-03-2013

DOI: 10.1371/JOURNAL.PONE.0058298

Structural characterization of a novel monotreme-specific protein with antimicrobial activity from the milk of the platypus

Publisher: International Union of Crystallography (IUCr)

Date: 2018

DOI: 10.1107/S2053230X17017708

Abstract: Monotreme lactation protein (MLP) is a recently identified protein with antimicrobial activity. It is present in the milk of monotremes and is unique to this lineage. To characterize MLP and to gain insight into the potential role of this protein in the evolution of lactation, the crystal structure of duck-billed platypus ( Ornithorhynchus anatinus ) MLP was determined at 1.82 Å resolution. This is the first structure to be reported for this novel, mammalian antibacterial protein. MLP was expressed as a FLAG epitope-tagged protein in mammalian cells and crystallized readily, with at least three space groups being observed ( P 1, C 2 and P 2 1 ). A 1.82 Å resolution native data set was collected from a crystal in space group P 1, with unit-cell parameters a = 51.2, b = 59.7, c = 63.1 Å, α = 80.15, β = 82.98, γ = 89.27°. The structure was solved by SAD phasing using a protein crystal derivatized with mercury in space group C 2, with unit-cell parameters a = 92.7, b = 73.2, c = 56.5 Å, β = 90.28°. MLP comprises a monomer of 12 helices and two short β-strands, with much of the N-terminus composed of loop regions. The crystal structure of MLP reveals no three-dimensional similarity to any known structures and reveals a heretofore unseen fold, supporting the idea that monotremes may be a rich source for the identification of novel proteins. It is hypothesized that MLP in monotreme milk has evolved to specifically support the unusual lactation strategy of this lineage and may have played a central role in the evolution of these mammals.

Structural and Functional Basis of Resistance to Neuraminidase Inhibitors of Influenza B Viruses

Publisher: American Chemical Society (ACS)

Date: 09-08-2010

DOI: 10.1021/JM100621S

Design, Synthesis, and X-ray of Selenides as New Class of Agents for Prevention of Diabetic Cerebrovascular Pathology

Publisher: American Chemical Society (ACS)

Date: 09-04-2018

DOI: 10.1021/ACSMEDCHEMLETT.8B00076

What's in a Name? Moving Towards a Limited Vocabulary for Macromolecular Crystallisation

Publisher: CSIRO Publishing

Date: 2014

DOI: 10.1071/CH14199

Abstract: This paper discusses the need for a systematic and standard naming nomenclature within the field of macromolecular crystallisation, and presents a set of rules and standard names which provides a start towards this end. The field of protein crystallisation is populated by biologists and chemists, and the dictionary in use needs to be unambiguous to both disciplines, yet must have useability as the most fundamental tenet if it is going to be widely adopted.

Intermolecular cleavage by UmuD-like enzymes: identification of residues required for cleavage and substrate specificity 1 1Edited by A. Gottesman

Publisher: Elsevier BV

Date: 02-1999

DOI: 10.1006/JMBI.1998.2433

Abstract: The UmuD-like proteins are best characterized for their role in damage-induced SOS mutagenesis. An essential step in this process is the enzymatic self-processing of the UmuD-like proteins. This reaction is thought to occur either via an intramolecular or intermolecular self-cleavage mechanism. Here, we demonstrate that it can also occur via an heterologous intermolecular cleavage reaction. The Escherichia coli UmuD enzyme demonstrated the broadest substrate specificity, cleaving both E. coli and Salmonella typhimurium UmuD substrates in vivo. In comparison, the wild-type S. typhimurium UmuD (UmuDSt) and MucA enzymes catalyzed intermolecular self-cleavage, but did not facilitate heterologous cleavage. Heterologous cleavage by the UmuDSt enzyme was, however, observed with chimeric UmuD substrates that possess residues 30-55 of UmuDSt. We have further localized the residue predominantly responsible for UmuDSt-catalyzed heterologous cleavage to Ser50 in the substrate molecule. We hypothesize that changes at this residue affect the positioning of the cleavage site of a substrate molecule within the catalytic cleft of the UmuDSt enzyme by affecting the formation of a so-called UmuD "filament-dimer". This hypothesis is further supported by the observation that mutations known to disrupt an E. coli UmuD' filament dimer also block intermolecular UmuDEc cleavage.

The C6 Web Tool: A Resource for the Rational Selection of Crystallization Conditions

Publisher: American Chemical Society (ACS)

Date: 10-05-2010

DOI: 10.1021/CG1004209

Fragment Screening for the Modelling Community: SPR, ITC, and Crystallography

Publisher: CSIRO Publishing

Date: 2013

DOI: 10.1071/CH13302

Abstract: The SAMPL (Statistical Assessment of the Modelling of Proteins and Ligands) challenge brought together experimentalists and modellers in an effort to improve our understanding of chemical and biochemical systems so better modelling tools can be developed. The most recent challenge, SAMPL3, held at Stanford University in August 2011, was an attempt to improve the methods used to predict how small fragment compounds bind to proteins, and the protein chosen for this test was bovine trypsin. Surface plasmon resonance was used to screen 500 compounds from a Maybridge fragment library and these compounds were subsequently used to soak crystals of trypsin and the best hits were also characterised by isothermal titration calorimetry. We present methods used for the surface plasmon resonance and the isothermal titration calorimetry experiments, as well as the results for these methods and those compounds that were found in the crystal structures.

300-Fold Increase in Production of the Zn ²⁺ -Dependent Dechlorinase TrzN in Soluble Form via Apoenzyme Stabilization

Publisher: American Society for Microbiology

Date: 07-2014

DOI: 10.1128/AEM.00916-14

Abstract: Microbial metalloenzymes constitute a large library of biocatalysts, a number of which have already been shown to catalyze the breakdown of toxic chemicals or industrially relevant chemical transformations. However, while there is considerable interest in harnessing these catalysts for biotechnology, for many of the enzymes, their large-scale production in active, soluble form in recombinant systems is a significant barrier to their use. In this work, we demonstrate that as few as three mutations can result in a 300-fold increase in the expression of soluble TrzN, an enzyme from Arthrobacter aurescens with environmental applications that catalyzes the hydrolysis of triazine herbicides, in Escherichia coli . Using a combination of X-ray crystallography, kinetic analysis, and computational simulation, we show that the majority of the improvement in expression is due to stabilization of the apoenzyme rather than the metal ion-bound holoenzyme. This provides a structural and mechanistic explanation for the observation that many compensatory mutations can increase levels of soluble-protein production without increasing the stability of the final, active form of the enzyme. This study provides a molecular understanding of the importance of the stability of metal ion free states to the accumulation of soluble protein and shows that differences between apoenzyme and holoenzyme structures can result in mutations affecting the stability of either state differently.

Benzensulfonamides bearing spyrohydantoin moieties act as potent inhibitors of human carbonic anhydrases II and VII and show neuropathic pain attenuating effects

Publisher: Elsevier BV

Date: 09-2019

DOI: 10.1016/J.EJMECH.2019.05.058

Abstract: Carbonic Anhydrases have been recently validated as novel therapeutic targets in neuropathic pain. In this study, we combine the anticonvulsant propriety of spyrohydantoin and the CA inhibitor moiety of benzenesulfonamide to synthesize a novel series of spyrohydantoin bearing sulfonamides with strong activity against hCA II and VII. These isoforms are present in the nervous system and largely expressed both at the central as well as at peripheral level and can be modulated for pain relief. The crystal structures of hCA II in complex with selected compounds 5a-c demonstrate the importance of the tail in the binding modes within the isoform. Finally, in vivo, in an animal model of oxaliplatin induced neuropathy, compounds with organoselenium tails (8b-c) showed potent neuropathic pain attenuating effects. Taken together, these data strongly suggest the translational utility of these inhibitors as novel pain relievers.

Identification of a highly conserved allosteric binding site on Mnk1 and Mnk2

Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)

Date: 12-08-2015

DOI: 10.1124/MOL.115.100131

Abstract: Elevated levels of phosphorylated eukaryotic initiation factor 4E (eIF4E) have been implicated in many tumor types, and mitogen activated protein kinase-interacting kinases (Mnks) are the only known kinases that phosphorylate eIF4E at Ser209. The phosphorylation of eIF4E is essential for oncogenic transformation but is of no significance to normal growth and development. Pharmacological inhibition of Mnks therefore provides a nontoxic and effective strategy for cancer therapy. However, a lack of specific Mnk inhibitors has confounded pharmacological target validation and clinical development. Herein, we report the identification of a novel series of Mnk inhibitors and their binding modes. A systematic workflow has been established to distinguish between type III and type I/II inhibitors. A selection of 66 compounds was tested for Mnk1 and Mnk2 inhibition, and 9 out of 20 active compounds showed type III interaction with an allosteric site of the proteins. Most of the type III inhibitors exhibited dual Mnk1 and Mnk2 activities and demonstrated potent antiproliferative properties against the MV4-11 acute myeloid leukemia cell line. Interestingly, ATP-/substrate-competitive inhibitors were found to be highly selective for Mnk2, with little or no activity for Mnk1. Our study suggests that Mnk1 and Mnk2 share a common structure of the allosteric inhibitory binding site but possess different structural features of the ATP catalytic domain. The findings will assist in the future design and development of Mnk targeted anticancer therapeutics.

The X-ray crystal structure of the N-terminal domain of Ssr4, a Schizosaccharomyces pombe chromatin-remodelling protein

Publisher: International Union of Crystallography (IUCr)

Date: 25-11-2020

DOI: 10.1107/S2053230X20015216

Abstract: Ssr4 is a yeast protein from Schizosaccharomyces pombe and is an essential part of the chromatin-remodelling [SWI/SNF and RSC (remodelling the structure of chromatin)] complexes found in S. pombe . These complexes (or their homologues) regulate gene expression in eukaryotic organisms, affecting a large number of genes both positively and negatively. The downstream effects are seen in development, and in humans have implications for disease such as cancer. The chromatin structure is altered by modifying the DNA–histone contacts, thus opening up or closing down sections of DNA to specific transcription factors that regulate the transcription of genes. The Ssr4 sequence has little homology to other sequences in the Protein Data Bank, so the structure was solved using an iodine derivative with SAD phasing. The structure of the N-terminal domain is an antiparallel β-sheet of seven strands with α-helices on one side and random coil on the other. The structure is significantly different to deposited structures and was used as a target in the most recent Critical Assessment of Techniques for Protein Structure Prediction (CASP predictioncenter.org/) competition.

A double-emulsion microfluidic platform forin vitrogreen fluorescent protein expression

Publisher: IOP Publishing

Date: 28-04-2011

DOI: 10.1088/0960-1317/21/5/054032

An unexpected vestigial protein complex reveals the evolutionary origins of an s-triazine catabolic enzyme

Publisher: Elsevier BV

Date: 05-2018

DOI: 10.1074/JBC.RA118.001996

High-Resolution X-Ray Structures of Two Functionally Distinct Members of the Cyclic Amide Hydrolase Family of Toblerone Fold Enzymes

Publisher: American Society for Microbiology

Date: 05-2017

DOI: 10.1128/AEM.03365-16

Abstract: The Toblerone fold was discovered recently when the first structure of the cyclic amide hydrolase, AtzD (a cyanuric acid hydrolase), was elucidated. We surveyed the cyclic amide hydrolase family, finding a strong correlation between phylogenetic distribution and specificity for either cyanuric acid or barbituric acid. One of six classes (IV) could not be tested due to a lack of expression of the proteins from it, and another class (V) had neither cyanuric acid nor barbituric acid hydrolase activity. High-resolution X-ray structures were obtained for a class VI barbituric acid hydrolase (1.7 Å) from a Rhodococcus species and a class V cyclic amide hydrolase (2.4 Å) from a Frankia species for which we were unable to identify a substrate. Both structures were homologous with the tetrameric Toblerone fold enzyme AtzD, demonstrating a high degree of structural conservation within the cyclic amide hydrolase family. The barbituric acid hydrolase structure did not contain zinc, in contrast with early reports of zinc-dependent activity for this enzyme. Instead, each barbituric acid hydrolase monomer contained either Na + or Mg 2+ , analogous to the structural metal found in cyanuric acid hydrolase. The Frankia cyclic amide hydrolase contained no metal but instead formed unusual, reversible, intermolecular vicinal disulfide bonds that contributed to the thermal stability of the protein. The active sites were largely conserved between the three enzymes, differing at six positions, which likely determine substrate specificity. IMPORTANCE The Toblerone fold enzymes catalyze an unusual ring-opening hydrolysis with cyclic amide substrates. A survey of these enzymes shows that there is a good correlation between physiological function and phylogenetic distribution within this family of enzymes and provide insights into the evolutionary relationships between the cyanuric acid and barbituric acid hydrolases. This family of enzymes is structurally and mechanistically distinct from other enzyme families however, to date the structure of just two, physiologically identical, enzymes from this family has been described. We present two new structures: a barbituric acid hydrolase and an enzyme of unknown function. These structures confirm that members of the CyAH family have the unusual Toblerone fold, albeit with some significant differences.

Some of the most interesting CASP11 targets through the eyes of their authors

Publisher: Wiley

Date: 16-11-2015

DOI: 10.1002/PROT.24942

Crystal Structures of Novel Allosteric Peptide Inhibitors of HIV Integrase Identify New Interactions at the LEDGF Binding Site

Publisher: Wiley

Date: 17-08-2011

DOI: 10.1002/CBIC.201100350

Abstract: An optimised method of solution cyclisation gave us access to a series of peptides including SLKIDNLD (2). We investigated the crystallographic complexes of the HIV integrase (HIV-IN) catalytic core domain with 13 of the peptides and identified multiple interactions at the binding site, including hydrogen bonds with residues Thr125 and Gln95, that have not previously been described as being accessible within the binding site. We show that the peptides inhibit the interaction of lens epithelium-derived growth factor (LEDGF) with HIV-IN in a proximity AlphaScreen assay and in an assay for the LEDGF enhancement of HIV-IN strand transfer. The interactions identified represent a potential framework for the development of new HIV-IN inhibitors.

Design, synthesis and X-ray crystallography of selenides bearing benzenesulfonamide moiety with neuropathic pain modulating effects

Publisher: Elsevier BV

Date: 06-2018

DOI: 10.1016/J.EJMECH.2018.05.026

Abstract: A series of selenides bearing benzensulfonamide were investigated as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). Potent inhibitory action, in the low nanomolar range, was detected against isoforms hCA II and VII, which are known to be involved in neuropathic pain modulation. These selenides showed on the other hand moderate inhibition against the cytosolic isoforms hCA I and transmembrane hCA IX. X-ray crystallographic data of two derivatives bound to hCA II allowed us to rationalize the excellent inhibitory data. In a mice model of neuropathic pain induced by oxaliplatin, some of the strong CA II/VII inhibitors induced a long lasting pain relieving effect.

Nitrate in the active site of protein tyrosine phosphatase 1B is a putative mimetic of the transition state

Publisher: International Union of Crystallography (IUCr)

Date: 31-01-2014

DOI: 10.1107/S1399004713031052

Abstract: The X-ray crystal structure of the complex of protein tyrosine phosphatase 1B with nitrate anion has been determined and modelled quantum-mechanically. Two protomers were present in the structure, one with the mechanistically important WPD loop closed and the other with this loop open. Nitrate was observed bound to each protomer, making close contacts with the S atom of the catalytic cysteine and a tyrosine residue from a crystallographically related protomer.

Carbonic Anhydrase Inhibitors with Dual-Tail Moieties To Match the Hydrophobic and Hydrophilic Halves of the Carbonic Anhydrase Active Site

Publisher: American Chemical Society (ACS)

Date: 27-01-2015

DOI: 10.1021/JM501798G

Abstract: We present a new approach to carbonic anhydrase II (CA II) inhibitor design that enables close interrogation of the regions of the CA active site where there is the greatest variability in amino acid residues among the different CA isozymes. By appending dual tail groups onto the par excellence CA inhibitor acetazolamide, compounds that may interact with the distinct hydrophobic and hydrophilic halves of the CA II active site were prepared. The dual-tail combinations selected included (i) two hydrophobic moieties, (ii) two hydrophilic moieties, and (iii) one hydrophobic and one hydrophilic moiety. The CA enzyme inhibition profile as well as the protein X-ray crystal structure of compound 3, comprising one hydrophobic and one hydrophilic tail moiety, in complex with CA II is described. This novel dual-tail approach has provided an enhanced opportunity to more fully exploit interactions with the CA active site by enabling these molecules to interact with the distinct halves of the active site. In addition to the dual-tail compounds, a corresponding set of single-tail derivatives was synthesized, enabling a comparative analysis of the single-tail versus dual-tail compound CA inhibition profile.

Structural basis for the allosteric regulation of the SbtA bicarbonate transporter by the PII-like protein, SbtB, from Cyanobium sp. PCC7001

Publisher: Cold Spring Harbor Laboratory

Date: 09-09-2019

DOI: 10.1101/762807

Abstract: Cyanobacteria have evolved a suite of enzymes and inorganic carbon (C i ) transporters that improve photosynthetic performance by increasing the localized concentration of CO 2 around the primary CO 2 -fixating enzyme, Rubisco. This CO 2 -concentrating mechanism (CCM) is highly regulated, responds to illumination/darkness cycles and allows cyanobacteria to thrive under limiting C i conditions. While the transcriptional control of CCM activity is well understood, less is known about how regulatory proteins might allosterically regulate C i transporters in response to changing conditions. Cyanobacterial sodium-dependent bicarbonate transporters (SbtAs) are inhibited by P II -like regulatory proteins (SbtBs), with the inhibitory effect being modulated by adenylnucleotides. Here, we used isothermal titration calorimetry to show that SbtB from Cyanobium sp. PCC7001 (SbtB7001) binds AMP, ADP, cAMP and ATP with micromolar-range affinities. X-ray crystal structures of apo- and nucleotide-bound SbtB7001 revealed that while AMP, ADP and cAMP have little effect on the SbtB7001 structure, binding of ATP stabilizes the otherwise flexible T-loop and that the flexible C-terminal C-loop adopts several distinct conformations. We also show that ATP binding affinity is increased ten-fold in the presence of Ca 2+ and we present an X-ray crystal structure of Ca 2+ ATP:SbtB7001 that shows how this metal ion facilitates additional stabilizing interactions with the apex of the T-loop. We propose that the Ca 2+ ATP-induced conformational change observed in SbtB7001 is important for allosteric regulation of SbtA activity by SbtB and is consistent with changing adenylnucleotide levels in illumination/darkness cycles.

Production and crystallization of a selenomethionyl variant of UmuD', an Escherichia coli SOS response protein

Publisher: Wiley

Date: 08-1996

DOI: 10.1002/(SICI)1097-0134(199608)25:4<506::AID-PROT10>3.0.CO;2-L

Structural basis for ligand recognition by a Cache chemosensory domain that mediates carboxylate sensing in Pseudomonas syringae

Publisher: Springer Science and Business Media LLC

Date: 13-10-2016

DOI: 10.1038/SREP35198

Abstract: Chemoreceptors enable bacteria to detect chemical signals in the environment and navigate towards niches that are favourable for survival. The sensor domains of chemoreceptors function as the input modules for chemotaxis systems, and provide sensory specificity by binding specific ligands. Cache-like domains are the most common extracellular sensor module in prokaryotes, however only a handful have been functionally or structurally characterised. Here, we have characterised a chemoreceptor Cache-like sensor domain (PscD-SD) from the plant pathogen Pseudomonas syringae pv. actinidiae ( Psa ). High-throughput fluorescence thermal shift assays, combined with isothermal thermal titration calorimetry, revealed that PscD-SD binds specifically to C 2 (glycolate and acetate) and C 3 (propionate and pyruvate) carboxylates. We solved the structure of PscD-SD in complex with propionate using X-ray crystallography. The structure reveals the key residues that comprise the ligand binding pocket and dictate the specificity of this sensor domain for C 2 and C 3 carboxylates. We also demonstrate that all four carboxylate ligands are chemoattractants for Psa , but only two of these (acetate and pyruvate) are utilisable carbon sources. This result suggests that in addition to guiding the bacteria towards nutrients, another possible role for carboxylate sensing is in locating potential sites of entry into the host plant.

Correction: Small molecule inhibitors of the LEDGF site of human immunodeficiency virus integrase identified by fragment screening and structure based design (PLoS ONE)

Publisher: Public Library of Science (PLoS)

Date: 24-07-2013

DOI: 10.1371/ANNOTATION/EB238F0F-7582-4318-AF95-64AC98D3B0BE

From information management to protein annotation: Preparing protein structures for drug discovery

Publisher: International Union of Crystallography (IUCr)

Date: 21-10-2002

DOI: 10.1107/S0907444902016852

Abstract: In contrast to academic pursuits of structural genomics, Structural GenomiX (SGX) solves protein structures at high throughput for the main purpose of enhancing drug-discovery projects, either internally or in partnership with pharmaceutical/biotechnology companies. This involves a radical redesign of the pipeline of methods that turn a gene sequence into a three-dimensional protein structure. The various processes all report electronically to a Laboratory Information Management System (LIMS) to make sure all the parameters of the experiment are recorded in an accessible and `mineable' form, helping guarantee reproducibility of results. Quality control at several key points keeps the process from branching out on a wrong hypothesis. Protein annotation, in a broad sense, takes care of the interpretation of a protein crystal structure or the crystal structure of one or several protein–ligand complexes. This interpretation both gathers all necessary biological information (protein function, mechanism, specific features within a protein family etc .) and hands over this information in a form accessible to medicinal chemistry teams designing specific small-molecule agonists or antagonists.

An efficient high-throughput screening method for MYST family Acetyltransferases, a new class of epigenetic drug targets

Publisher: Elsevier BV

Date: 12-2011

DOI: 10.1177/1087057111421631

Abstract: Epigenetic aberrations are increasingly regarded as key factors in cancer progression. Recently, deregulation of histone acetyltransferases (HATs) has been linked to several types of cancer. Monocytic leukemia zinc finger protein (MOZ) is a member of the MYST family of HATs, which regulate gene expression in cell proliferation and differentiation. Deregulation of these processes through constitutively active MOZ fusion proteins gives rise to the formation of leukemic stem cells, rendering MOZ an excellent target for treating myeloid leukemia. The authors implemented a hit discovery c aign to identify small-molecule inhibitors of MOZ-HAT activity. They developed a robust, homogeneous assay measuring the acetylation of synthetic histone peptides. In a primary screening c aign testing 243 000 lead-like compounds, they identified inhibitors from several chemical classes. Secondary assays were used to eliminate assay-interfering compounds and prioritize confirmed hits. This study establishes a new high-throughput assay for HAT activity and could provide the foundation for the development of a new class of drugs for the treatment of leukemias.

Structural Basis for the Selective Binding of Inhibitors to 6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase from Staphylococcus aureus and Escherichia coli

Publisher: American Chemical Society (ACS)

Date: 26-04-2016

DOI: 10.1021/ACS.JMEDCHEM.6B00002

Abstract: 6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is a member of the folate biosynthesis pathway found in prokaryotes and lower eukaryotes that catalyzes the pyrophosphoryl transfer from the ATP cofactor to a 6-hydroxymethyl-7,8-dihydropterin substrate. We report the chemical synthesis of a series of S-functionalized 8-mercaptoguanine (8MG) analogues as substrate site inhibitors of HPPK and quantify binding against the E. coli and S. aureus enzymes (EcHPPK and SaHPPK). The results demonstrate that analogues incorporating acetophenone-based substituents have comparable affinities for both enzymes. Preferential binding of benzyl-substituted 8MG derivatives to SaHPPK was reconciled when a cryptic pocket unique to SaHPPK was revealed by X-ray crystallography. Differential chemical shift perturbation analysis confirmed this to be a common mode of binding for this series to SaHPPK. One compound (41) displayed binding affinities of 120 nM and 1.76 μM for SaHPPK and EcHPPK, respectively, and represents a lead for the development of more potent and selective inhibitors of SaHPPK.

Unexpected Isomerisation of a Fragment Analogue During Fragment-Based Screening of HIV Integrase Catalytic Core Domain

Publisher: CSIRO Publishing

Date: 2015

DOI: 10.1071/CH15587

Abstract: Fragment-based screening of human immunodeficiency virus type 1 (HIV) integrase revealed several aromatic carboxylic acid fragment hits, some of which bound weakly at the site on the HIV-integrase catalytic core domain that binds the lens epithelium-derived growth factor (LEDGF). Virtual screening of an internal database identified an analogue that bound with higher affinity and in an isomerised form to the LEDGF binding site. The starting lactone was stable in CDCl3 however, an unexpected isomerisation process occurred in [D6]DMSO to give the same isomer found in the LEDGF binding site. This hit led directly to a series of low-micromolar LEDGF inhibitors and, via a scaffold hop, to a series of allosteric binding site inhibitors.

Cyclic Secondary Sulfonamides: Unusually Good Inhibitors of Cancer-Related Carbonic Anhydrase Enzymes

Publisher: American Chemical Society (ACS)

Date: 11-04-2014

DOI: 10.1021/JM500255Y

Abstract: Carbonic anhydrase IX (CA IX) is a target for hypoxic cancer therapies, and the discovery of CA IX selective ligands is imperative for the development of these agents. Primary sulfonamides are broad specificity inhibitors of CA enzymes, while secondary sulfonamides are generally poor CA inhibitors. However, saccharin, a cyclic secondary sulfonamide, has unusually good inhibition of CA IX (Ki = 103 nM). In this study, we demonstrate that the affinity and selectivity of saccharin for CA IX can be further modulated when linked to hydrophobic or hydrophilic substituents. The hydrophilic glycoconjugate derivative (12) showed improved inhibition of CA IX (Ki = 49.5 nM) and extremely poor inhibition of the predominant off-target CAs (Ki > 50000 nM) compared to saccharin. This >1000-fold selectivity for CA IX over off-target CAs is unprecedented for classical primary sulfonamide CA inhibitors. Our study highlights the potential of cyclic secondary sulfonamides to be exploited for the discovery of potent, cancer-selective CA inhibitors.

The 1.59 Å Resolution Crystal Structure of TM0096, a Flavin Mononucleotide Binding Protein from Thermotoga maritima

Publisher: Wiley

Date: 05-03-2004

DOI: 10.1002/PROT.20086

Crystal Structure of YIGZ, a Conserved Hypothetical Protein from Escherichia coli K12 with a Novel Fold

Publisher: Wiley

Date: 14-04-2004

DOI: 10.1002/PROT.20087

Construction of the CSIRO Fragment Library

Publisher: CSIRO Publishing

Date: 2013

DOI: 10.1071/CH13325

Abstract: A fundamental component of a successful fragment screening program is a productive fragment library, one that delivers hit fragments with potential for pharmaceutical development. A proprietary fragment library was developed by identifying and extracting subsets of CSIRO’s Compound Collection using two complimentary approaches. Over time, the use of surface plasmon resonance as a front-line screening tool has enabled identification and exclusion of problematic compounds and led to a more reliable fragment screening library.

Inhibitors of histone acetyltransferases KAT6A/B induce senescence and arrest tumour growth

Publisher: Springer Science and Business Media LLC

Date: 08-2018

DOI: 10.1038/S41586-018-0387-5

Crystallization and preliminary X-ray diffraction analysis of the amidase domain of allophanate hydrolase from Pseudomonas sp strain ADP

Publisher: International Union of Crystallography (IUCr)

Date: 19-02-2014

DOI: 10.1107/S2053230X13034705

Synthesis of novel tellurides bearing benzensulfonamide moiety as carbonic anhydrase inhibitors with antitumor activity

Publisher: Elsevier BV

Date: 11-2019

DOI: 10.1016/J.EJMECH.2019.111586

Abstract: We have synthetized a novel series of β-hydroxy tellurides bearing the benzenesulfonamide group as potent inhibitors of carbonic anhydrase enzymes. In a one pot procedure, we discovered both the ring opening reaction of the three-membered ring and the cleavage of the sulfonamide protecting moiety at the same time. Moreover, the first X-ray co-crystallographic structure of a β-hydroxy telluride derivative with hCA II is reported. The potent effects of these compounds against the tumor-associated hCA IX with low nanomolar constant inhibition values give the possibility to evaluate their activity in vitro using a breast cancer cell line (MDA-MB-231). Compounds 7e and 7g induced significant toxic effects against tumor cells after 48 h incubation in normoxic conditions killing over 50% of tumor cells at 3 μM, but their efficacy decreased in hypoxic conditions reaching the 50% of the tumor cell viability only at 30 μM. These unusual features make them interesting lead compounds to act as antitumor agents, not only as Carbonic Anhydrase IX inhibitors, but reasonably in different pathways, where hCA IX is not overexpressed.

Enzyme synthesis and activity assay in microfluidic droplets on a chip

Publisher: Wiley

Date: 04-2011

DOI: 10.1002/ELSC.201000043

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.

Crystal structures of human ENPP1 in apo and bound forms

Publisher: International Union of Crystallography (IUCr)

Date: 17-08-2020

DOI: 10.1107/S2059798320010505

Abstract: Cancer is one of the leading causes of mortality in humans, and recent work has focused on the area of immuno-oncology, in which the immune system is used to specifically target cancerous cells. Ectonucleotide pyrophosphatase hosphodiesterase 1 (ENPP1) is an emerging therapeutic target in human cancers owing to its role in degrading cyclic GMP-AMP (cGAMP), an agonist of the stimulator of interferon genes (STING). The available structures of ENPP1 are of the mouse enzyme, and no structures are available with anything other than native nucleotides. Here, the first X-ray crystal structures of the human ENPP1 enzyme in an apo form, with bound nucleotides and with two known inhibitors are presented. The availability of these structures and a robust crystallization system will allow the development of structure-based drug-design c aigns against this attractive cancer therapeutic target.

A novel decarboxylating amidohydrolase involved in avoiding metabolic dead ends during cyanuric acid catabolism in Pseudomonas sp. strain ADP

Publisher: Public Library of Science (PLoS)

Date: 06-11-2018

DOI: 10.1371/JOURNAL.PONE.0206949

Over the rainbow: structural characterisation of the chromoproteins gfasPurple, amilCP, spisPink and eforRed

Publisher: International Union of Crystallography (IUCr)

Date: 08-04-2022

DOI: 10.1107/S2059798322002625

Abstract: Anthozoan chromoproteins are highly pigmented, ersely coloured and readily produced in recombinant expression systems. While they are a versatile and powerful building block in synthetic biology for applications such as biosensor development, they are not widely used in comparison to the related fluorescent proteins, partly due to a lack of structural characterization to aid protein engineering. Here, high-resolution X-ray crystal structures of four open-source chromoproteins, gfasPurple, amilCP, spisPink and eforRed, are presented. These proteins are dimers in solution, and mutation at the conserved dimer interface leads to loss of visible colour development in gfasPurple. The chromophores are trans and noncoplanar in gfasPurple, amilCP and spisPink, while that in eforRed is cis and noncoplanar, and also emits fluorescence. Like other characterized chromoproteins, gfasPurple, amilCP and eforRed contain an sp 2 -hybridized N -acylimine in the peptide bond preceding the chromophore, while spisPink is unusual and demonstrates a true sp 3 -hybridized trans -peptide bond at this position. It was found that point mutations at the chromophore-binding site in gfasPurple that substitute similar amino acids to those in amilCP and spisPink generate similar colours. These features and observations have implications for the utility of these chromoproteins in protein engineering and synthetic biology applications.

A β-alanine catabolism pathway containing a highly promiscuous ω-transaminase in the 12-aminododecanate-degrading Pseudomonas sp. strain AAC

Publisher: American Society for Microbiology

Date: 07-2016

DOI: 10.1128/AEM.00665-16

Abstract: We previously isolated the transaminase KES23458 from Pseudomonas sp. strain AAC as a promising biocatalyst for the production of 12-aminododecanoic acid, a constituent building block of nylon-12. Here, we report the subsequent characterization of this transaminase. It exhibits activity with a broad substrate range which includes α-, β-, and ω-amino acids, as well as α,ω-diamines and a number of other industrially relevant compounds. It is therefore a prospective candidate for the biosynthesis of a range of polyamide monomers. The crystal structure of KES23458 revealed that the protein forms a dimer containing a large active site pocket and unusual phosphorylated histidine residues. To infer the physiological role of the transaminase, we expressed, purified, and characterized a dehydrogenase from the same operon, KES23460. Unlike the transaminase, the dehydrogenase was shown to be quite selective, catalyzing the oxidation of malonic acid semialdehyde, formed from β-alanine transamination via KES23458. In keeping with previous reports, the dehydrogenase was shown to catalyze both a coenzyme A (CoA)-dependent reaction to form acetyl-CoA and a significantly slower CoA-independent reaction to form acetate. These findings support the original functional assignment of KES23458 as a β-alanine transaminase. However, a seemingly well-adapted active site and promiscuity toward unnatural compounds, such as 12-aminododecanoic acid, suggest that this enzyme could perform multiple functions for Pseudomonas sp. strain AAC. IMPORTANCE We describe the characterization of an industrially relevant transaminase able to metabolize 12-aminododecanoic acid, a constituent building block of the widely used polymer nylon-12, and we report the biochemical and structural characterization of the transaminase protein. A physiological role for this highly promiscuous enzyme is proposed based on the characterization of a related gene from the host organism. Molecular dynamics simulations were carried out to compare the conformational changes in the transaminase protein to better understand the determinants of specificity in the protein. This study makes a substantial contribution that is of interest to the broad biotechnology and enzymology communities, providing insights into the catalytic activity of an industrially relevant biocatalyst as well as the biological function of this operon.

Fragment screening for a protein-protein interaction inhibitor to WDR5.

Publisher: AIP Publishing

Date: 11-2019

DOI: 10.1063/1.5122849

Abstract: The WD40-repeat protein WDR5 scaffolds various epigenetic writers and is a critical component of the mammalian SET/MLL histone methyltransferase complex. Dysregulation of the MLL1 catalytic function is associated with mixed-lineage leukemia, and antagonism of the WDR5-MLL1 interaction by small molecules has been proposed as a therapeutic strategy for MLL-rearranged cancers. Small molecule binders of the “WIN” site of WDR5 that cause displacement from chromatin have been additionally implicated to be of broader use in cancer treatment. In this study, a fragment screen with Surface Plasmon Resonance (SPR) was used to identify a highly ligand-efficient imidazole-containing compound that is bound in the WIN site. The subsequent medicinal chemistry c aign—guided by a suite of high-resolution cocrystal structures with WDR5—progressed the initial hit to a low micromolar binder. One outcome from this study is a moiety that substitutes well for the side chain of arginine a tripeptide containing one such substitution was resolved in a high resolution structure (1.5 Å) with a binding mode analogous to the native tripeptide. SPR furthermore indicates a similar residence time (kd = ∼0.06 s−1) for these two analogs. This novel scaffold therefore represents a possible means to overcome the potential permeability issues of WDR5 ligands that possess highly basic groups like guanidine. The series reported here furthers the understanding of the WDR5 WIN site and functions as a starting point for the development of more potent WDR5 inhibitors that may serve as cancer therapeutics.

X-Ray structure and mutagenesis studies of the n-isopropylammelide isopropylaminohydrolase, AtzC

Publisher: Public Library of Science (PLoS)

Date: 21-09-2015

DOI: 10.1371/JOURNAL.PONE.0137700

Characterization of crystals of the thermostable DNA polymerase I from Thermus aquaticus

Publisher: Wiley

Date: 09-1995

DOI: 10.1002/PROT.340230112

Abstract: Thermus aquaticus DNA polymerase I is an enzyme that is of both physiological and technological interest. It carries out template-directed polymerization of DNA at elevated temperatures and is widely used in polymerase chain reaction (PCR). We have obtained crystals of the enzyme that diffracts X-rays to at least 3.0 A resolution in a cubic space group. Determination of the three-dimensional structure of the native enzyme along with those of relevant complexes will greatly enhance our knowledge of molecular events involved in DNA replication, will permit improvements in PCR, and will add to our knowledge of the structural bases of thermostability in proteins.

The Glitazone Class of Drugs as Carbonic Anhydrase Inhibitors—A Spin-Off Discovery from Fragment Screening

Publisher: MDPI AG

Date: 18-05-2021

DOI: 10.3390/MOLECULES26103010

Abstract: The approved drugs that target carbonic anhydrases (CA, EC 4.2.1.1), a family of zinc metalloenzymes, comprise almost exclusively of primary sulfonamides (R-SO2NH2) as the zinc binding chemotype. New clinical applications for CA inhibitors, particularly for hard-to-treat cancers, has driven a growing interest in the development of novel CA inhibitors. We recently discovered that the thiazolidinedione heterocycle, where the ring nitrogen carries no substituent, is a new zinc binding group and an alternate CA inhibitor chemotype. This heterocycle is curiously also a substructure of the glitazone class of drugs used in the treatment options for type 2 diabetes. Herein, we investigate and characterise three glitazone drugs (troglitazone 11, rosiglitazone 12 and pioglitazone 13) for binding to CA using native mass spectrometry, protein X-ray crystallography and hydrogen–deuterium exchange (HDX) mass spectrometry, followed by CA enzyme inhibition studies. The glitazone drugs all displayed appreciable binding to and inhibition of CA isozymes. Given that thiazolidinediones are not credited as a zinc binding group nor known as CA inhibitors, our findings indicate that CA may be an off-target of these compounds when used clinically. Furthermore, thiazolidinediones may represent a new opportunity for the development of novel CA inhibitors as future drugs.

Privileged scaffolds in medicinal chemistry: Studies on pyrazolo[1,5-a]pyrimidines on sulfonamide containing Carbonic Anhydrase inhibitors

Publisher: Elsevier BV

Date: 10-2021

DOI: 10.1016/J.BMCL.2021.128309

Abstract: We report for the first time a small series of compounds endowed in vitro with inhibitory properties for the human (h) expressed Carbonic Anhydrase (CAs, E.C. 4.2.1.1) enzymes of physiological interest (i.e. I, II, VA, IX and XII) and bearing the pyrazolo[1,5-a]pyrimidine (PP) scaffold at the tail section. Among the series reported, 1a-3a, 7a, 8a, 1b and 2b resulted effective ligands and with good selectivities for the hCAs II, IX or XII. In consideration of the nearly matching K

Structural and biochemical characterization of the biuret hydrolase (BiuH) from the cyanuric acid catabolism pathway of Rhizobium leguminasorum bv. viciae 3841

Publisher: Public Library of Science (PLoS)

Date: 09-02-2018

DOI: 10.1371/JOURNAL.PONE.0192736

Crystallization of an apo form of human arginase: Using all the tools in the toolbox simultaneously

Publisher: International Union of Crystallography (IUCr)

Date: 23-12-2011

DOI: 10.1107/S1744309110046208

A drunken search in crystallization space

Publisher: International Union of Crystallography (IUCr)

Date: 30-09-2014

DOI: 10.1107/S2053230X1401841X

Abstract: The REMARK280 field of the Protein Data Bank is the richest open source of successful crystallization information. The REMARK280 field is optional and currently uncurated, so significant effort needs to be applied to extract reliable data. There are well over 15 000 crystallization conditions available commercially from 12 different vendors. After putting the PDB crystallization information and the commercial cocktail data into a consistent format, these data are used to extract information about the overlap between the two sets of crystallization conditions. An estimation is made as to which commercially available conditions are most appropriate for producing well diffracting crystals by looking at which commercial conditions are found unchanged (or almost unchanged) in the PDB. Further analyses include which commercial kits are the most appropriate for shotgun or more traditional approaches to crystallization screening. This analysis suggests that almost 40% of the crystallization conditions found currently in the PDB are identical or very similar to a commercial condition.

Solution structure of Pyrobaculum aerophilum DsrC, an archaeal homologue of the gamma subunit of dissimilatory sulfite reductase

Publisher: Wiley

Date: 15-11-2001

DOI: 10.1046/J.0014-2956.2001.02529.X

Abstract: The solution structure of DsrC, an archaeal homologue of the gamma subunit of dissimilatory sulfite reductase, has been determined by NMR spectroscopy. This 12.7-kDa protein from the hyperthermophilic archaeon Pyrobaculum aerophilum adopts a novel fold consisting of an orthogonal helical bundle with a beta hairpin along one side. A portion of the structure resembles the helix-turn-helix DNA-binding motif common in transcriptional regulator proteins. The protein contains two disulfide bonds but remains folded following reduction of the disulfides. DsrC proteins from organisms other than Pyrobaculum species do not contain these disulfide bonds. A conserved cysteine next to the C-terminus, which is not involved in the disulfide bonds, is located on a seven-residue C-terminal arm that is not part of the globular protein and is likely to dynamically s le more than one conformation.

X-ray crystal structure of a malonate-semialdehyde dehydrogenase from Pseudomonas sp strain AAC

Publisher: International Union of Crystallography (IUCr)

Date: 2017

DOI: 10.1107/S2053230X16020008

Abstract: The NAD-dependent malonate-semialdehyde dehydrogenase KES23460 from Pseudomonas sp. strain AAC makes up half of a bicistronic operon responsible for β-alanine catabolism to produce acetyl-CoA. The KES23460 protein has been heterologously expressed, purified and used to generate crystals suitable for X-ray diffraction studies. The crystals belonged to space group P 2 1 2 1 2 1 and diffracted X-rays to beyond 3 Å resolution using the microfocus beamline of the Australian Synchrotron. The structure was solved using molecular replacement, with a monomer from PDB entry 4zz7 as the search model.

Crystal structure of a putrescine aminotransferase from Pseudomonas sp strain AAC

Publisher: International Union of Crystallography (IUCr)

Date: 2017

DOI: 10.1107/S2053230X16019658

Abstract: The putrescine aminotransferase KES24511 from Pseudomonas sp. strain AAC was previously identified as an industrially relevant enzyme based on the discovery that it is able to promiscuously catalyse the transamination of 12-aminododecanoic acid. Here, the cloning, heterologous expression, purification and successful crystallization of the KES24511 protein are reported, which ultimately generated crystals adopting space group I 2. The crystals diffracted X-rays to 2.07 Å resolution and data were collected using the microfocus beamline of the Australian Synchrotron. The structure was solved using molecular replacement, with a monomer from PDB entry 4a6t as the search model.

Discovery of Potent Dual-Tailed Benzenesulfonamide Inhibitors of Human Carbonic Anhydrases Implicated in Glaucoma and in Vivo Profiling of Their Intraocular Pressure-Lowering Action

Publisher: American Chemical Society (ACS)

Date: 07-02-2020

DOI: 10.1021/ACS.JMEDCHEM.9B02090

Identification of a New Zinc Binding Chemotype by Fragment Screening

Publisher: American Chemical Society (ACS)

Date: 09-2017

DOI: 10.1021/ACS.JMEDCHEM.7B00606

Abstract: The discovery of a new zinc binding chemotype from screening a nonbiased fragment library is reported. Using the orthogonal fragment screening methods of native state mass spectrometry and surface plasmon resonance a 3-unsubstituted 2,4-oxazolidinedione fragment was found to have low micromolar binding affinity to the zinc metalloenzyme carbonic anhydrase II (CA II). This affinity approached that of fragment sized primary benzenesulfonamides, the classical zinc binding group found in most CA II inhibitors. Protein X-ray crystallography established that 3-unsubstituted 2,4-oxazolidinediones bound to CA II via an interaction of the acidic ring nitrogen with the CA II active site zinc, as well as two hydrogen bonds between the oxazolidinedione ring oxygen and the CA II protein backbone. Furthermore, 3-unsubstituted 2,4-oxazolidinediones appear to be a viable starting point for the development of an alternative class of CA inhibitor, wherein the medicinal chemistry pedigree of primary sulfonamides has dominated for several decades.

Discovery of New Selenoureido Analogues of 4-(4-Fluorophenylureido)benzenesulfonamide as Carbonic Anhydrase Inhibitors

Publisher: American Chemical Society (ACS)

Date: 14-08-2017

DOI: 10.1021/ACSMEDCHEMLETT.7B00280

Crystallization and preliminary X-ray analysis of the complexes between a Fab and two forms of human insulin-like growth factor II

Publisher: International Union of Crystallography (IUCr)

Date: 26-08-2009

DOI: 10.1107/S1744309109024932

The structure of vanin 1: A key enzyme linking metabolic disease and inflammation

Publisher: International Union of Crystallography (IUCr)

Date: 28-11-2014

DOI: 10.1107/S1399004714022767

Abstract: Although part of the coenzyme A pathway, vanin 1 (also known as pantetheinase) sits on the cell surface of many cell types as an ectoenzyme, catalyzing the breakdown of pantetheine to pantothenic acid (vitamin B 5 ) and cysteamine, a strong reducing agent. Vanin 1 was initially discovered as a protein involved in the homing of leukocytes to the thymus. Numerous studies have shown that vanin 1 is involved in inflammation, and more recent studies have shown a key role in metabolic disease. Here, the X-ray crystal structure of human vanin 1 at 2.25 Å resolution is presented, which is the first reported structure from the vanin family, as well as a crystal structure of vanin 1 bound to a specific inhibitor. These structures illuminate how vanin 1 can mediate its biological roles by way of both enzymatic activity and protein–protein interactions. Furthermore, it sheds light on how the enzymatic activity is regulated by a novel allosteric mechanism at a domain interface.

The structure of the hexameric atrazine chlorohydrolase AtzA

Publisher: International Union of Crystallography (IUCr)

Date: 26-02-2015

DOI: 10.1107/S1399004715000619

Abstract: Atrazine chlorohydrolase (AtzA) was discovered and purified in the early 1990s from soil that had been exposed to the widely used herbicide atrazine. It was subsequently found that this enzyme catalyzes the first and necessary step in the breakdown of atrazine by the soil organism Pseudomonas sp. strain ADP. Although it has taken 20 years, a crystal structure of the full hexameric form of AtzA has now been obtained. AtzA is less well adapted to its physiological role ( i.e. atrazine dechlorination) than the alternative metal-dependent atrazine chlorohydrolase (TrzN), with a substrate-binding pocket that is under considerable strain and for which the substrate is a poor fit.

Structures of the transcriptional regulator BgaR, a lactose sensor

Publisher: International Union of Crystallography (IUCr)

Date: 26-06-2019

DOI: 10.1107/S2059798319008131

Abstract: The structure of BgaR, a transcriptional regulator of the lactose operon in Clostridium perfringens , has been solved by SAD phasing using a mercury derivative. BgaR is an exquisite sensor of lactose, with a binding affinity in the low-micromolar range. This sensor and regulator has been captured bound to lactose and to lactulose as well as in a nominal apo form, and was compared with AraC, another saccharide-binding transcriptional regulator. It is shown that the saccharides bind in the N-terminal region of a jelly-roll fold, but that part of the saccharide is exposed to bulk solvent. This differs from the classical AraC saccharide-binding site, which is mostly sequestered from the bulk solvent. The structures of BgaR bound to lactose and to lactulose highlight how specific and nonspecific interactions lead to a higher binding affinity of BgaR for lactose compared with lactulose. Moreover, solving multiple structures of BgaR in different space groups, both bound to saccharides and unbound, verified that the dimer interface along a C-terminal helix is similar to the dimer interface observed in AraC.

Practical Aspects of the SAMPL Challenge: Providing an Extensive Experimental Data Set for the Modeling Community

Publisher: Elsevier BV

Date: 12-2009

DOI: 10.1177/1087057109348220

Abstract: To provide an experimental basis for a comprehensive molecular modeling evaluation study, 500 fragments from the Maybridge fragment library were soaked into crystals of bovine pancreatic trypsin and the structures determined by X-ray crystallography. The soaking experiments were performed in both single and pooled aliquots to determine if combination of fragments is an appropriate strategy. A further set of data was obtained from co-crystallizing the pooled fragments with the protein. X-ray diffraction data were collected on approximately 1000 crystals at the Australian Synchrotron, and these data were subsequently processed, and the preliminary analysis was performed with a custom software application (Jigsaw), which combines available software packages for structure solution and analysis.

How to fabricate robust microfluidic systems for a dollar

Publisher: SPIE

Date: 07-12-2013

DOI: 10.1117/12.2033508

Crystal structure of an indole-3-acetic acid amido synthetase from grapevine involved in auxin homeostasis

Publisher: Oxford University Press (OUP)

Date: 11-2012

DOI: 10.1105/TPC.112.102921

Haloalkane Dehalogenases:  Structure of a Rhodococcus Enzyme^,

Publisher: American Chemical Society (ACS)

Date: 12-11-1999

DOI: 10.1021/BI9913855

Abstract: The hydrolytic haloalkane dehalogenases are promising bioremediation and biocatalytic agents. Two general classes of dehalogenases have been reported from Xanthobacter and Rhodococcus. While these enzymes share 30% amino acid sequence identity, they have significantly different substrate specificities and halide-binding properties. We report the 1.5 A resolution crystal structure of the Rhodococcus dehalogenase at pH 5.5, pH 7.0, and pH 5.5 in the presence of NaI. The Rhodococcus and Xanthobacter enzymes have significant structural homology in the alpha/beta hydrolase core, but differ considerably in the cap domain. Consistent with its broad specificity for primary, secondary, and cyclic haloalkanes, the Rhodococcus enzyme has a substantially larger active site cavity. Significantly, the Rhodococcus dehalogenase has a different catalytic triad topology than the Xanthobacter enzyme. In the Xanthobacter dehalogenase, the third carboxylate functionality in the triad is provided by D260, which is positioned on the loop between beta7 and the penultimate helix. The carboxylate functionality in the Rhodococcus catalytic triad is donated from E141. A model of the enzyme cocrystallized with sodium iodide shows two iodide binding sites one that defines the normal substrate and product-binding site and a second within the active site region. In the substrate and product complexes, the halogen binds to the Xanthobacter enzyme via hydrogen bonds with the N(eta)H of both W125 and W175. The Rhodococcusenzyme does not have a tryptophan analogous to W175. Instead, bound halide is stabilized with hydrogen bonds to the N(eta)H of W118 and to N(delta)H of N52. It appears that when cocrystallized with NaI the Rhodococcus enzyme has a rare stable S-I covalent bond to S(gamma) of C187.

Cyanuric acid hydrolase: evolutionary innovation by structural concatenation

Publisher: Wiley

Date: 20-05-2013

DOI: 10.1111/MMI.12249

Cryo-EM structure of the active, Gs-protein complexed, human CGRP receptor

Publisher: Springer Science and Business Media LLC

Date: 09-2018

DOI: 10.1038/S41586-018-0535-Y

Design of a methotrexate-controlled chemical dimerization system and its use in bio-electronic devices

Publisher: Springer Science and Business Media LLC

Date: 08-12-2021

DOI: 10.1038/S41467-021-27184-W

Abstract: Natural evolution produced polypeptides that selectively recognize chemical entities and their polymers, ranging from ions to proteins and nucleic acids. Such selective interactions serve as entry points to biological signaling and metabolic pathways. The ability to engineer artificial versions of such entry points is a key goal of synthetic biology, bioengineering and bioelectronics. We set out to map the optimal strategy for developing artificial small molecule:protein complexes that function as chemically induced dimerization (CID) systems. Using several starting points, we evolved CID systems controlled by a therapeutic drug methotrexate. Biophysical and structural analysis of methotrexate-controlled CID system reveals the critical role played by drug-induced conformational change in ligand-controlled protein complex assembly. We demonstrate utility of the developed CID by constructing electrochemical biosensors of methotrexate that enable quantification of methotrexate in human serum. Furthermore, using the methotrexate and functionally related biosensor of rapamycin we developed a multiplexed bioelectronic system that can perform repeated measurements of multiple analytes. The presented results open the door for construction of genetically encoded signaling systems for use in bioelectronics and diagnostics, as well as metabolic and signaling network engineering.

Azidothymidine “Clicked” into 1,2,3-Triazoles: First Report on Carbonic Anhydrase–Telomerase Dual-Hybrid Inhibitors

Publisher: American Chemical Society (ACS)

Date: 28-05-2020

DOI: 10.1021/ACS.JMEDCHEM.0C00636

Small Molecule Inhibitors of the LEDGF Site of Human Immunodeficiency Virus Integrase Identified by Fragment Screening and Structure Based Design

Publisher: Public Library of Science (PLoS)

Date: 10-07-2012

DOI: 10.1371/JOURNAL.PONE.0040147

8‐Mercaptoguanine Derivatives as Inhibitors of Dihydropteroate Synthase

Publisher: Wiley

Date: 09-01-2018

DOI: 10.1002/CHEM.201704730

Abstract: Dihydropteroate synthase (DHPS) is an enzyme of the folate biosynthesis pathway, which catalyzes the formation of 7,8-dihydropteroate (DHPt) from 6-hydroxymethyl-7,8-dihydropterin pyrophosphate (DHPPP) and para-aminobenzoic acid (pABA). DHPS is the long-standing target of the sulfonamide class of antibiotics that compete with pABA. In the wake of sulfa drug resistance, targeting the structurally rigid (and more conserved) pterin site has been proposed as an alternate strategy to inhibit DHPS in wild-type and sulfa drug resistant strains. Following the work on developing pterin-site inhibitors of the adjacent enzyme 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), we now present derivatives of 8-mercaptoguanine, a fragment that binds weakly within both enzymes, and quantify sub-μm binding using surface plasmon resonance (SPR) to Escherichia coli DHPS (EcDHPS). Eleven ligand-bound EcDHPS crystal structures delineate the structure-activity relationship observed providing a structural framework for the rational development of novel, substrate-envelope-compliant DHPS inhibitors.

Interrogating HIV integrase for compounds that bind- a SAMPL challenge

Publisher: Springer Science and Business Media LLC

Date: 16-02-2014

DOI: 10.1007/S10822-014-9721-7

Understanding the control of grape berry ripening and developing opportunities for its manipulation

Publisher: International Society for Horticultural Science (ISHS)

Date: 04-2017

DOI: 10.17660/ACTAHORTIC.2017.1157.1

A nondenaturing purification scheme for the DNA-binding domain of poly(ADP-ribose) polymerase, a structure-specific DNA-binding protein

Publisher: Elsevier BV

Date: 10-1998

DOI: 10.1006/PREP.1998.0919

Abstract: Poly(ADP-ribose) polymerase (PARP) is thought to be involved in DNA repair given its ability to recognize and bind to DNA strand breaks. During apoptosis, PARP is proteolytically cleaved into two stable fragments, the N-terminal 25-kDa DNA-binding domain (DBD) and the 85-kDa fragment containing the automodification and catalytic domains. To understand the DNA-binding properties of PARP, we expressed a recombinant hexahistidine tagged protein (His-DBD) in Escherichia coli. We modified expression to facilitate protein folding by including zinc and reducing the induction temperature. Properly folded, the DNA-binding domain of PARP binds to single- and double-stranded DNA in a structure-specific manner. To eliminate contamination with bacterial DNA that occurred during the purification process, a purification procedure was developed to produce DNA-free protein. In addition, to remove the hexahistidine tag from the recombinant protein, thrombin cleavage was carried out while the recombinant protein was bound to a DNA column. This procedure stabilized the recombinant protein and resulted in nearly 100% cleavage with no appreciable loss to unwanted proteolytic degradation. This nondenaturing purification scheme results in high-quality, native PARP-DBD for use in structural and biochemical studies.

Phoenito experiments: Combining the strengths of commercial crystallization automation

Publisher: International Union of Crystallography (IUCr)

Date: 31-10-2008

DOI: 10.1107/S1744309108029667

Native State Mass Spectrometry, Surface Plasmon Resonance, and X-ray Crystallography Correlate Strongly as a Fragment Screening Combination

Publisher: American Chemical Society (ACS)

Date: 29-02-2016

DOI: 10.1021/ACS.JMEDCHEM.5B01940

Abstract: Fragment-based drug discovery (FBDD) is contingent on the development of analytical methods to identify weak protein-fragment noncovalent interactions. Herein we have combined an underutilized fragment screening method, native state mass spectrometry, together with two proven and popular fragment screening methods, surface plasmon resonance and X-ray crystallography, in a fragment screening c aign against human carbonic anhydrase II (CA II). In an initial fragment screen against a 720-member fragment library (the "CSIRO Fragment Library") seven CA II binding fragments, including a selection of nonclassical CA II binding chemotypes, were identified. A further 70 compounds that comprised the initial hit chemotypes were subsequently sourced from the full CSIRO compound collection and screened. The fragment results were extremely well correlated across the three methods. Our findings demonstrate that there is a tremendous opportunity to apply native state mass spectrometry as a complementary fragment screening method to accelerate drug discovery.

Rational engineering of a mesohalophilic carbonic anhydrase to an extreme halotolerant biocatalyst

Publisher: Springer Science and Business Media LLC

Date: 21-12-2015

DOI: 10.1038/NCOMMS10278

Abstract: Enzymes expressed by highly salt-tolerant organisms show many modifications compared with salt-affected counterparts including biased amino acid and lower α-helix content, lower solvent accessibility and negative surface charge. Here, we show that halotolerance can be generated in an enzyme solely by modifying surface residues. Rational design of carbonic anhydrase II is undertaken in three stages replacing 18 residues in total, crystal structures confirm changes are confined to surface residues. Catalytic activities and thermal unfolding temperatures of the designed enzymes increase at high salt concentrations demonstrating their shift to halotolerance, whereas the opposite response is found in the wild-type enzyme. Molecular dynamics calculations reveal a key role for sodium ions in increasing halotolerant enzyme stability largely through interactions with the highly ordered first Na + hydration shell. For the first time, an approach to generate extreme halotolerance, a trait with broad application in industrial biocatalysis, in a wild-type enzyme is demonstrated.

Integrated microdroplet-based system for enzyme synthesis and sampling

Publisher: SPIE

Date: 07-12-2013

DOI: 10.1117/12.2033569

T-dependent B cell responses to Plasmodium induce antibodies that form a high-avidity multivalent complex with the circumsporozoite protein

Publisher: Public Library of Science (PLoS)

Date: 31-07-2017

DOI: 10.1371/JOURNAL.PPAT.1006469

SGX Pharmaceuticals

Location: United States of America

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University Of New South Wales

Location: Australia

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CSIRO Biomedical Manufacturing

Location: Australia

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OpenEye Scientific Software Inc

Location: United States of America

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Linkage Infrastructure, Equipment And Facilities - Grant ID: LE210100122

Start Date: 2021

End Date: 12-2021

Amount: $725,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP140101495

Start Date: 2014

End Date: 12-2017

Amount: $390,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE170100075

Start Date: 2017

End Date: 12-2017

Amount: $315,000.00

Funder: Australian Research Council