Luke Guddat

Measuring teacher self-efficacy

Publisher: Frontiers Media SA

Date: 24-01-2023

DOI: 10.3389/FPSYG.2022.1063830

Abstract: Challenges exist in the validating procedure and comprehensiveness of the existing TSE measurements, though advancements have been achieved. Also, less consistencies have been received regarding teacher self-efficacy measurement in Chinese context so that the study developed and validated a new comprehensive scale for this construct. A total of 854 Chinese pre-service teachers responded to 40 purposely selected teacher self-efficacy items, together with the Generalized Self-Efficacy Scale, the agreeableness subscale of the Big Five Inventory, and items on their effectiveness of teaching practicing and intention to be a teacher. Exploratory factor analyses revealed two distinct factors, one factor (Ethos) focused on the general school climate, harmony, and cooperation, as well as teachers’ own professional development, the other (Teaching) focused on aspects of classrooms and student learning. Confirmatory and second-order factor analysis supported the existence of two factors and also indicated one overarching construct of teacher self-efficacy. Both domains were significantly correlated with general self-efficacy and agreeableness, with either moderate or low correlations. Significant differences in teacher self-efficacy for Ethos and Teaching were found between pre-service teachers who reported higher levels of effectiveness during their professional placement and greater intention to be teachers compared to those with lower self-ratings. In addition, a 20-item short version of the scale was developed, and the same factorial structure was confirmed. This study validated the two-factor structure of a newly developed teacher self-efficacy scale that covers domains both within and outside classroom teaching. Limitations and implications are discussed.

Structural aspects of conformational changes in ligand binding by antibody fragments

Publisher: Elsevier BV

Date: 1994

DOI: 10.1016/S0923-2494(94)80045-6

Herbicide-binding sites revealed in the structure of plant acetohydroxyacid synthase

Publisher: Proceedings of the National Academy of Sciences

Date: 10-01-2006

DOI: 10.1073/PNAS.0508701103

Abstract: The sulfonylureas and imidazolinones are potent commercial herbicide families. They are among the most popular choices for farmers worldwide, because they are nontoxic to animals and highly selective. These herbicides inhibit branched-chain amino acid biosynthesis in plants by targeting acetohydroxyacid synthase (AHAS, EC 2.2.1.6). This report describes the 3D structure of Arabidopsis thaliana AHAS in complex with five sulfonylureas (to 2.5 Å resolution) and with the imidazolinone, imazaquin (IQ 2.8 Å). Neither class of molecule has a structure that mimics the substrates for the enzyme, but both inhibit by blocking a channel through which access to the active site is gained. The sulfonylureas approach within 5 Å of the catalytic center, which is the C2 atom of the cofactor thiamin diphosphate, whereas IQ is at least 7 Å from this atom. Ten of the amino acid residues that bind the sulfonylureas also bind IQ. Six additional residues interact only with the sulfonylureas, whereas there are two residues that bind IQ but not the sulfonylureas. Thus, the two classes of inhibitor occupy partially overlapping sites but adopt different modes of binding. The increasing emergence of resistant weeds due to the appearance of mutations that interfere with the inhibition of AHAS is now a worldwide problem. The structures described here provide a rational molecular basis for understanding these mutations, thus allowing more sophisticated AHAS inhibitors to be developed. There is no previously described structure for any plant protein in complex with a commercial herbicide.

An unusual human IgM antibody with a protruding HCDR3 and high avidity for its peptide ligands

Publisher: Elsevier BV

Date: 04-2000

DOI: 10.1016/S0161-5890(00)00049-3

Abstract: The crystal structure of the Fv molecule from a human monoclonal IgM cryoglobulin (Mez) was determined at 2.6 A resolution. Amino acid sequences of framework regions (FR) of the Mez light (L) and heavy (H) chain variable domains (VL and VH) are highly similar to their counterparts in another human Fv (Pot) previously subjected to X-ray analysis in our laboratory. As expected, the three-dimensional (3-D) structures of FR are quite similar in the two proteins, as are four of the six complementarity-determining regions (CDRs): CDRs 1 and 2 for both L and H chains. Absence of Pro 95L from the LCDR3 loop in Mez VL (relative to Pot LCDR3) results in compression of this loop and creates more space in the VL-VH interface. In the two IgMs, HCDR3 conformations differ significantly from all previously defined conformations for these loops. Pot has a 12-residue HCDR3 that collapses to fill all available space in the VL-VH domain interface, resulting in the formation of a relatively flat platform for antigen binding. In Mez, the HCDR3 is two residues longer and is comprehensively different. A semi-rigid ascending segment dominated by a Pro-Pro-Tyr sequence protrudes out into solvent. The descending portion has the sequence Gly-Trp-Gly-Gly-Gly, which promotes high local flexibility. This segment folds across the VL-VH domain interface to interact with residues in LCDR3. These features partition the Mez active site into two compartments, a large cavity between VL and VH and a smaller cavity lined entirely by constituents of the three heavy chain CDRs. Such an unusual topographical feature indicates why the Mez IgM does not bind to the Fc portion of intact human IgG antibodies in immunoassays yet interacts with high avidity with many Fc-derived octapeptides. The cavities are expected to be the repositories for the Fc-derived peptides, while the semi-rigid protrusion of the Mez HCDR3 prevents the close approach of another macromolecule (e.g. intact IgG) to the active site.

Structural basis of resistance to herbicides that target acetohydroxyacid synthase

Publisher: Springer Science and Business Media LLC

Date: 11-06-2022

DOI: 10.1038/S41467-022-31023-X

Abstract: Acetohydroxyacid synthase (AHAS) is the target for more than 50 commercial herbicides first applied to crops in the 1980s. Since then, 197 site-of-action resistance isolates have been identified in weeds, with mutations at P197 and W574 the most prevalent. Consequently, AHAS is at risk of not being a useful target for crop protection. To develop new herbicides, a functional understanding to explain the effect these mutations have on activity is required. Here, we show that these mutations can have two effects (i) to reduce binding affinity of the herbicides and (ii) to abolish time-dependent accumulative inhibition, critical to the exceptional effectiveness of this class of herbicide. In the two mutants, conformational changes occur resulting in a loss of accumulative inhibition by most herbicides. However, bispyribac, a bulky herbicide is able to counteract the detrimental effects of these mutations, explaining why no site-of-action resistance has yet been reported for this herbicide.

Role of Human Hypoxanthine Guanine Phosphoribosyltransferase in Activation of the Antiviral Agent T-705 (Favipiravir)

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

Date: 08-2013

DOI: 10.1124/MOL.113.087247

Abstract: 6-Fluoro-3-hydroxy-2-pyrazinecarboxamide (T-705) is a novel antiviral compound with broad activity against influenza virus and erse RNA viruses. Its active metabolite, T-705-ribose-5'-triphosphate (T-705-RTP), is recognized by influenza virus RNA polymerase as a substrate competing with GTP, giving inhibition of viral RNA synthesis and lethal virus mutagenesis. Which enzymes perform the activation of T-705 is unknown. We here demonstrate that human hypoxanthine guanine phosphoribosyltransferase (HGPRT) converts T-705 into its ribose-5'-monophosphate (RMP) prior to formation of T-705-RTP. The anti-influenza virus activity of T-705 and T-1105 (3-hydroxy-2-pyrazinecarboxamide the analog lacking the 6-fluoro atom) was lost in HGPRT-deficient Madin-Darby canine kidney cells. This HGPRT dependency was confirmed in human embryonic kidney 293T cells undergoing HGPRT-specific gene knockdown followed by influenza virus ribonucleoprotein reconstitution. Knockdown for adenine phosphoribosyltransferase (APRT) or nicotinamide phosphoribosyltransferase did not change the antiviral activity of T-705 and T-1105. Enzymatic assays showed that T-705 and T-1105 are poor substrates for human HGPRT having Km(app) values of 6.4 and 4.1 mM, respectively. Formation of the RMP metabolites by APRT was negligible, and so was the formation of the ribosylated metabolites by human purine nucleoside phosphorylase. Phosphoribosylation and antiviral activity of the 2-pyrazinecarboxamide derivatives was shown to require the presence of the 3-hydroxyl but not the 6-fluoro substituent. The crystal structure of T-705-RMP in complex with human HGPRT showed how this compound binds in the active site. Since conversion of T-705 by HGPRT appears to be inefficient, T-705-RMP prodrugs may be designed to increase the antiviral potency of this new antiviral agent.

Stereo-Defined Acyclic Nucleoside Phosphonates are Selective and Potent Inhibitors of Parasite 6-Oxopurine Phosphoribosyltransferases

Publisher: American Chemical Society (ACS)

Date: 17-02-2022

DOI: 10.1021/ACS.JMEDCHEM.1C01881

Abstract: Pathogens such as

Local and Transmitted Conformational Changes on Complexation of an Anti-sweetener Fab

Publisher: Elsevier BV

Date: 02-1994

DOI: 10.1006/JMBI.1994.1133

Abstract: Crystal structures of an Fab (NC6.8) from a murine IgG2b(kappa) antibody and its complex with a sweet-tasting, N-,N'-,N"-trisubstituted guanidine compound (NC174) have been determined by X-ray analysis. Both crystal forms are produced by a microseeding technique in polyethylene glycol (PEG) 8000 but the habits and space groups are very different. The native protein crystallizes as plates in the monoclinic space group C2 and the complex crystallizes as prisms in the orthorhombic space group P2(1)2(1)2. The structures were solved by molecular replacement methods, with the Fab fragments from the 4-4-20, HyHel-5 and BV04-01 antibodies as starting models. On binding of the ligand, N-(p-cyanophenyl)-N'-(diphenylmethyl)-N"-(carboxymethyl)g uan idine, the protein exhibits significant local conformational changes in the active site, particularly in the third complementarity-determining region (CDR3) of the heavy chain. The ligand enters the small crevice by end-on insertion with the cyanophenyl group in the lead and the diphenyl rings partially protruding from the entrance. No strict pi-pi stacking interactions are observed. However, tyrosine L32 (CDR1), tyrosine L96 (CDR3) and tryptophan H33 (CDR1) help immobilize the cyanophenyl ring and guanido group, and tyrosine H96 moves about 4.5 A to lie between the rings of the diphenyl group. The positive charge on the guanido group is compensated by glutamic acid H50 (CDR2) while the negative charge on acetic acid is neutralized by arginine H56 (CDR2) and by hydrogen bonding with asparagine H58 (CDR2). Water molecules participate in the binding process by hydrogen bonding with the cyano and guanido groups. The mechanism of binding is a clear ex le of induced fit. Like hemoglobin, the NC6.8 Fab can be classified as an allosteric protein, since its overall structure is altered by the binding of a small ligand. In crystals of the native Fab the elbow bend angle is 184 degrees while in crystals of the complex the elbow angle is 153 degrees. There is also a reciprocal push-pull type of change where the heavy chain is flexed and the light chain is extended. The tail of the heavy chain, which would be connected to the Fc in an intact antibody, is displaced 19 A relative to its position in the unliganded Fab. Within the limited series of sweetener-Fab complexes we have thus far examined, only the NC174 hapten has produced such results.(ABSTRACT TRUNCATED AT 400 WORDS)

Crystallization of the FAD-independent acetolactate synthase ofKlebsiella pneumoniae

Publisher: International Union of Crystallography (IUCr)

Date: 20-06-2002

DOI: 10.1107/S0907444902008132

Abstract: Leucine and valine are formed in a common pathway from pyruvate in which the first intermediate is 2-acetolactate. In some bacteria, this compound also has a catabolic fate as the starting point for the butanediol fermentation. The enzyme (EC 4.1.3.18) that forms 2-acetolactate is known as either acetohydroxyacid synthase (AHAS) or acetolactate synthase (ALS), with the latter name preferred for the catabolic enzyme. A significant difference between AHAS and ALS is that the former requires FAD for catalytic activity, although the reason for this requirement is not well understood. Both enzymes require the cofactor thiamine diphosphate. Here, the crystallization and preliminary X-ray diffraction analysis of the Klebsiella pneumoniae ALS is reported. Data to 2.6 A resolution have been collected at 100 K using a rotating-anode generator and an R-AXIS IV++ detector. Crystals have unit-cell parameters a = 137.4, b = 143.9, c = 134.4 A, alpha = 90, beta = 108.4, gamma = 90 degrees and belong to space group C2. Preliminary analysis indicates that there are four monomers located in each asymmetric unit.

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

Structural insights as to how iron-loaded siderophores are imported into Mycobacterium tuberculosis by IrtAB

Publisher: Cold Spring Harbor Laboratory

Date: 11-01-2022

DOI: 10.1101/2022.01.11.475891

Abstract: The ATP-binding cassette (ABC) transporter, IrtAB, plays a vital role in the replication and viability of Mycobacterium tuberculosis ( Mtb ), where its function is to import iron-loaded siderophores. Unusually, it adopts the typical fold of canonical ABC exporters. Here, we report the structure of unliganded Mtb IrtAB and its structure in complex with ATP, ADP, and an ATP analogue (AMP-PNP) at resolutions from 2.9 to 3.5 Å. In the ATP-bound state, the two nucleotide-binding domains (NBDs) form a “head-to-tail” dimer, but IrtAB has an unexpectedly occluded conformation, with the inner core forming a large hydrophilic cavity of about 4600 Å 3 . Comparison of the structure of the transporter in inward-facing and occluded conformations reveals that the NBD and the intracellular helical region of transmembrane domain (TMD) have an asymmetric allosteric mechanism when ATP binding/hydrolysis such that the one exhibits rigid-body rotation and the other moves in a concerted response as a rigid body. This study provides a molecular basis for the ATP-driven conformational changes that occur in IrtAB and an explanation as to how iron-loaded siderophores are imported into Mtb by IrtAB.

Inhibition of the Escherichia coli 6-Oxopurine Phosphoribosyltransferases by Nucleoside Phosphonates: Potential for New Antibacterial Agents

Publisher: American Chemical Society (ACS)

Date: 27-08-2013

DOI: 10.1021/JM400779N

Abstract: Escherichia coli (Ec) cells possess two purine salvage enzymes: xanthine-guanine phosphoribosyltransferase (XGPRT) and hypoxanthine phosphoribosyltransferase (HPRT). EcXGPRT shares a common structural feature with other members of this family, a flexible loop that closes over the active site during catalysis. The replacement of six of these amino acids by alanine has no effect on the Km for the two substrates. However, the Ki for the nucleoside monophosphate increases by 27-fold, and the kcat is reduced by ∼200-fold. Nucleoside phosphonates (NP) are good inhibitors of EcXGPRT and EcHPRT, with Ki values as low as 10 nM. In the absence of the flexible loop, these values increase by 5- to 30-fold, indicating the importance of the loop for high-affinity inhibition. Crystal structures of two NPs in complex with EcXGPRT explain the tight binding. Prodrugs of NPs with low Ki values for EcXGPRT or EcHPRT exhibit IC50 values between 5 and 23 μM against Mycobacterium tuberculosis in cell-based assays, suggesting that these compounds are therapeutic leads against pathogenic bacteria.

Probing the mechanism of the bifunctional enzyme ketol‐acid reductoisomerase by site‐directed mutagenesis of the active site

Publisher: Wiley

Date: 23-12-2004

DOI: 10.1111/J.1742-4658.2004.04506.X

Abstract: Ketol-acid reductoisomerase (EC 1.1.1.86) is involved in the biosynthesis of the branched-chain amino acids. It is a bifunctional enzyme that catalyzes two quite different reactions at a common active site an isomerization consisting of an alkyl migration, followed by an NADPH-dependent reduction of a 2-ketoacid. The 2-ketoacid formed by the alkyl migration is not released. Using the pure recombinant Escherichia coli enzyme, we show that the isomerization reaction has a highly unfavourable equilibrium constant. The reductase activity is shown to be relatively nonspecific and is capable of utilizing a variety of 2-ketoacids. The active site of the enzyme contains eight conserved polar amino acids and we have mutated each of these in order to dissect their contributions to the isomerase and reductase activities. Several mutations result in loss of the isomerase activity with retention of reductase activity. However, none of the 17 mutants examined have the isomerase activity only. We suggest a reason for this, involving direct reduction of a transition state formed during the isomerization, which is necessitated by the unfavourable equilibrium position of the isomerization. Our mechanism explains why the two activities must occur in a single active site without release of a 2-ketoacid and provides a rationale for the requirement for NADPH by the isomerase.

Changes in pre-service teacher self-efficacy for teaching in relation to professional experience placements

Publisher: SAGE Publications

Date: 03-01-2022

DOI: 10.1177/00049441211060474

Abstract: This article aims to contribute new, longitudinal evidence on teacher self-efficacy (TSE) by investigating changes in TSE over the last 2 years of an Australian initial teacher education program. Two hundred and one pre-service teachers were surveyed at three timepoints: (1) after the first professional experience placement, (2) before and (3) after the final placement, using the Scale for Teacher Self-Efficacy. Data were analysed using multilevel modelling. TSE for the domains of classroom management and student engagement decreased significantly between the first and before the commencement of the last professional experience placements. All three dimensions of TSE – instructional strategies, student engagement and classroom management – increased significantly during the final placement.

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

Publisher: Elsevier BV

Date: 11-2000

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

Identification of mammalian-like purple acid phosphatases in a wide range of plants

Publisher: Elsevier BV

Date: 05-2000

DOI: 10.1016/S0378-1119(00)00186-4

Abstract: Purple acid phosphatases (PAPs) comprise a family of binuclear metal-containing hydrolases, members of which have been isolated from plants, mammals and fungi. Polypeptide chains differ in size (animal approximately 35kDa, plant approximately 55kDa) and exhibit low sequence homology between kingdoms but all residues involved in co-ordination of the metal ions are invariant. A search of genomic databases was undertaken using a sequence pattern which includes the conserved residues. Several novel potential PAP sequences were detected, including the first known ex les from bacterial sources. Ten plant ESTs were also identified which, although possessing the conserved sequence pattern, were not homologous throughout their sequences to previously known plant PAPs. Based on these EST sequences, novel cDNAs from sweet potato, soybean, red kidney bean and Arabidopsis thaliana were cloned and sequenced. These sequences are more closely related to mammalian PAP than to previously characterized plant enzymes. Their predicted secondary structure is similar to that of the mammalian enzyme. A model of the sweet potato enzyme was generated based on the coordinates of pig PAP. These observations strongly suggest that the cloned cDNA sequences represent a second group of plant PAPs with properties more similar to the mammalian enzymes than to the high molecular weight plant enzymes.

The three-dimensional structure of a complex of a murine Fab (NC10.14) with a potent sweetener (NC174): an illustration of structural diversity in antigen recognition by immunoglobulins

Publisher: Elsevier BV

Date: 09-2000

DOI: 10.1006/JMBI.2000.4083

Commercial Herbicides Can Trigger the Oxidative Inactivation of Acetohydroxyacid Synthase

Publisher: Wiley

Date: 29-02-2016

DOI: 10.1002/ANIE.201511985

Abstract: Acetohydroxyacid synthase (AHAS) inhibitors are highly successful commercial herbicides. New kinetic data show that the binding of these compounds leads to reversible accumulative inhibition of AHAS. Crystallographic data (to a resolution of 2.17 Å) for an AHAS-herbicide complex shows that closure of the active site occurs when the herbicidal inhibitor binds, thus preventing exchange with solvent. This feature combined with new kinetic data shows that molecular oxygen promotes an accumulative inhibition leading to the conclusion that the exceptional potency of these herbicides is augmented by subversion of an inherent oxygenase side reaction. The reactive oxygen species produced by this reaction are trapped in the active site, triggering oxidation reactions that ultimately lead to the alteration of the redox state of the cofactor flavin adenine dinucleotide (FAD), a feature that accounts for the observed reversible accumulative inhibition.

Inhibition of hypoxanthine-guanine phosphoribosyltransferase by acyclic nucleoside phosphonates: a new class of antimalarial therapeutics.

Publisher: American Chemical Society (ACS)

Date: 15-06-2009

DOI: 10.1021/JM900267N

Abstract: The purine salvage enzyme hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) is essential for purine nucleotide and hence nucleic acid synthesis in the malaria parasite, Plasmodium falciparum. Acyclic nucleoside phosphonates (ANPs) are analogues of the nucleotide product of the reaction, comprising a purine base joined by a linker to a phosphonate moiety. K(i) values for 19 ANPs were determined for Pf HGXPRT and the corresponding human enzyme, HGPRT. Values for Pf HGXPRT were as low as 100 nM, with selectivity for the parasite enzyme of up to 58. Structures of human HGPRT in complex with three ANPs are reported. On binding, a large mobile loop in the free enzyme moves to partly cover the active site. For three ANPs, the IC(50) values for Pf grown in cell culture were 1, 14, and 46 microM, while the cytotoxic concentration for the first compound was 489 microM. These results provide a basis for the design of potent and selective ANP inhibitors of Pf HGXPRT as antimalarial drug leads.

Teachers’ understanding and use of mathematical structure

Publisher: Springer Science and Business Media LLC

Date: 20-06-2022

DOI: 10.1007/S13394-020-00342-X

Acyclic Nucleoside Phosphonates Containing a Second Phosphonate Group Are Potent Inhibitors of 6-Oxopurine Phosphoribosyltransferases and Have Antimalarial Activity

Publisher: American Chemical Society (ACS)

Date: 19-03-2013

DOI: 10.1021/JM301893B

Abstract: Acyclic nucleoside phosphonates (ANPs) that contain a 6-oxopurine base are good inhibitors of the Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) 6-oxopurine phosphoribosyltransferases (PRTs). Chemical modifications based on the crystal structure of 2-(phosphonoethoxy)ethylguanine (PEEG) in complex with human HGPRT have led to the design of new ANPs. These novel compounds contain a second phosphonate group attached to the ANP scaffold. {[(2-[(Guanine-9H-yl)methyl]propane-1,3-diyl)bis(oxy)]bis(methylene)}diphosphonic acid (compound 17) exhibited a Ki value of 30 nM for human HGPRT and 70 nM for Pf HGXPRT. The crystal structure of this compound in complex with human HGPRT shows that it fills or partially fills three critical locations in the active site: the binding sites of the purine base, the 5'-phosphate group, and pyrophosphate. This is the first HG(X)PRT inhibitor that has been able to achieve this result. Prodrugs have been synthesized resulting in IC50 values as low as 3.8 μM for Pf grown in cell culture, up to 25-fold lower compared to the parent compounds.

Crystal structures of a purple acid phosphatase, representing different steps of this enzyme's catalytic cycle

Publisher: Springer Science and Business Media LLC

Date: 2008

DOI: 10.1186/1472-6807-8-6

Crystal structures of some niobium and tantalum oxides

Publisher: Elsevier BV

Date: 02-1986

DOI: 10.1016/0022-4596(86)90020-4

HOROLOGIUM II: A SECOND ULTRA-FAINT MILKY WAY SATELLITE IN THE HOROLOGIUM CONSTELLATION

Publisher: American Astronomical Society

Date: 28-07-2015

DOI: 10.1088/2041-8205/808/2/L39

Three-dimensional structure of an Fv from a human IgM immunoglobulin

Publisher: Elsevier BV

Date: 11-1992

DOI: 10.1016/0022-2836(92)90500-J

Abstract: An IgM(kappa) immunoglobulin from a patient (Pot) with Waldenstrom's macroglobulinemia was hydrolyzed with pepsin to release a fragment consisting of the 'variable' (V) domains of the light and heavy chains plus eight residue 'tails' from the 'constant' (C) domains. The crystal structure of this fragment was determined at 2.3 A resolution by molecular replacement and crystallographic refinement methods. When examined separately, the light chain component closely resembles another human kappa chain (Rei) in both the beta-pleated sheet regions and the 'hypervariable' loops. The conserved pleated sheets in the heavy chain are similar to those in the human Kol IgG1 protein, but the third hypervariable loop in particular is different from that in any immunoglobulin structure described to date. As in the Kol protein, this loop blocks the access to any internal active site along the light-heavy chain interface. Unlike the Kol protein, however, the loop does not protrude beyond the boundaries of a conventional antigen combining site. Instead, it forms a very compact structure, which fills almost all residual space between the domains. This is an ex le of one dominant complementarity-determining region (CDR) essentially negating the ersity possible with five other CDRs in the two chains. Ordered water molecules are associated with light chain constituents along the interface, but not with CDR3 of the heavy chain. In screening exercises the Pot IgM failed to bind a wide variety of peptides. Together, the results suggest that ligand binding can only occur on external surfaces of the protein. These surfaces carry a limited number of side chains usually assigned to CDRs in more typical antibodies.

Three-dimensional structure of an immunoglobulin light-chain dimer with amyloidogenic properties

Publisher: International Union of Crystallography (IUCr)

Date: 26-04-2002

DOI: 10.1107/S0907444902004183

Abstract: The X-ray structure of an immunoglobulin light-chain dimer isolated from the urine as a "Bence-Jones protein" from a patient with multiple myeloma and amyloidosis (Sea) was determined at 1.94 A resolution and refined to R and R(free) factors of 0.22 and 0.25, respectively. This "amyloidogenic" protein crystallized in the orthorhombic P2(1)2(1)2(1) space group with unit-cell parameters a = 48.28, b = 83.32, c = 112.59 A as determined at 100 K. In the vital organs (heart and kidneys), the equivalent of the urinary protein produced fibrillar amyloid deposits which were fatal to the patient. Compared with the amyloidogenic Mcg light-chain dimer, the Sea protein was highly soluble in aqueous solutions and only crystallized at concentrations approaching 100 mg ml(-1). Both the Sea and Mcg proteins packed into crystals in highly ordered arrangements typical of strongly diffracting crystals of immunoglobulin fragments. Overall similarities and significant differences in the three-dimensional structures and crystalline properties are discussed for the Sea and Mcg Bence-Jones proteins, which together provide a generalized model of abnormalities present in lambda chains, facilitating a better understanding of amyloidosis of light-chain origin (AL).

Sulfonylureas Have Antifungal Activity and Are Potent Inhibitors of Candida albicans Acetohydroxyacid Synthase

Publisher: American Chemical Society (ACS)

Date: 31-12-2013

DOI: 10.1021/JM301501K

Abstract: The sulfonylurea herbicides exert their activity by inhibiting plant acetohydroxyacid synthase (AHAS), the first enzyme in the branched-chain amino acid biosynthesis pathway. It has previously been shown that if the gene for AHAS is deleted in Candida albicans , attenuation of virulence is achieved, suggesting AHAS as an antifungal drug target. Herein, we have cloned, expressed, and purified C. albicans AHAS and shown that several sulfonylureas are inhibitors of this enzyme and possess antifungal activity. The most potent of these compounds is ethyl 2-(N-((4-iodo-6-methoxypyrimidin-2-yl)carbamoyl)sulfamoyl)benzoate (10c), which has a K(i) value of 3.8 nM for C. albicans AHAS and an MIC₉₀ of 0.7 μg/mL for this fungus in cell-based assays. For the sulfonylureas tested there was a strong correlation between inhibitory activity toward C. albicans AHAS and fungicidal activity, supporting the hypothesis that AHAS is the target for their inhibitory activity within the cell.

Crystal structures of Trypanosoma brucei hypoxanthine – guanine – xanthine phosphoribosyltransferase in complex with IMP, GMP and XMP

Publisher: Wiley

Date: 24-07-2019

DOI: 10.1111/FEBS.14987

Abstract: The 6-oxopurine phosphoribosyltransferases (PRTs) are drug targets for the treatment of parasitic diseases. This is due to the fact that parasites are auxotrophic for the 6-oxopurine bases relying on salvage enzymes for the synthesis of their 6-oxopurine nucleoside monophosphates. In Trypanosoma brucei, the parasite that is the aetiological agent for sleeping sickness, there are three 6-oxopurine PRT isoforms. Two are specific for hypoxanthine and guanine, whilst the third, characterized here, uses all three naturally occurring bases with similar efficiency. Here, we have determined crystal structures for TbrHGXPRT in complex with GMP, XMP and IMP to investigate the structural basis for substrate specificity. The results show that Y201 and E208, not commonly observed within the purine binding pocket of 6-oxopurine PRTs, contribute to the versatility of this enzyme. The structures further show that a nearby water can act as an adaptor to facilitate the binding of XMP and GMP. When GMP binds, a water can accept a proton from the 2-amino group but when XMP binds, the equivalent water can donate its proton to the 2-oxo group. However, when IMP is bound, no water molecule is observed at that location. DATABASE: Coordinates and structure factors were submitted to the Protein Data Bank and have accession codes of 6MXB, 6MXC, 6MXD and 6MXG for the TbrHGXPRT.XMP complex, TbrHGXPRT.GMP complex, TbrHGXPRT.IMP complex, and TbrHGPRT.XMP complex, respectively.

Chemical Synthesis, in Vitro Acetohydroxyacid Synthase (AHAS) Inhibition, Herbicidal Activity, and Computational Studies of Isatin Derivatives

Publisher: American Chemical Society (ACS)

Date: 25-08-2011

DOI: 10.1021/JF2021607

Abstract: Acetohydroxyacid synthase (AHAS) catalyzes the first common step in the biosynthesis of the branched-chain amino acids. As a result of its metabolic importance in plants, it is a target for many commercial herbicides. Virtual screening analysis inspired the evaluation of 19 commercially available isatin analogues and 13 newly synthesized isatin derivatives as novel AHAS inhibitors and for their herbicidal activity. The best compound demonstrated 95% inhibition of the activity of Arabidopsis thaliana AHAS at a concentration of 100 mg L(-1), whereas the herbicidal activities of three compounds reached 50% inhibition at a concentration of 10 mg L(-1) using the rape root growth test. CoMFA contour models were established to understand the structure-activity relationships for this class of AHAS inhibitor. The compounds were docked to the active site cavity of A. thaliana AHAS using FlexX, and the dominant binding mode was consistent with frontier molecular orbital from DFT calculations. This is the first comprehensive study of isatin derivatives as AHAS inhibitors and provides a valuable starting point for the design of new herbicides.

Architecture of the mycobacterial succinate dehydrogenase with a membrane-embedded Rieske FeS cluster

Publisher: Proceedings of the National Academy of Sciences

Date: 05-04-2021

DOI: 10.1073/PNAS.2022308118

Abstract: Targeting energy metabolism in Mycobacterium tuberculosis has emerged as a new paradigm in antituberculosis drug discovery. Succinate dehydrogenase is considered the regulator of respiration in M. tuberculosis . Mycobacteria contains two different succinate dehydrogenase enzymes designated Sdh1 and Sdh2. Sdh1 has recently been identified as a new class of succinate dehydrogenase. In this study, we have determined M. smegmatis Sdh1 structures alone and in the presence of ubiquinone-1, revealing that Sdh1 has a novel electron transfer pathway and a unique substrate-binding site. These data show that the structure of M. tuberculosis Sdh1 is significantly different by comparison with the human counterpart making a good antituberculosis drug target.

Structure of the human respiratory complex II

Publisher: Proceedings of the National Academy of Sciences

Date: 25-04-2023

DOI: 10.1073/PNAS.2216713120

Abstract: Human complex II is a key protein complex that links two essential energy-producing processes: the tricarboxylic acid cycle and oxidative phosphorylation. Deficiencies due to mutagenesis have been shown to cause mitochondrial disease and some types of cancers. However, the structure of this complex is yet to be resolved, hindering a comprehensive understanding of the functional aspects of this molecular machine. Here, we have determined the structure of human complex II in the presence of ubiquinone at 2.86 Å resolution by cryoelectron microscopy, showing it comprises two water-soluble subunits, SDHA and SDHB, and two membrane-spanning subunits, SDHC and SDHD. This structure allows us to propose a route for electron transfer. In addition, clinically relevant mutations are mapped onto the structure. This mapping provides a molecular understanding to explain why these variants have the potential to produce disease.

The crystal structure of a bacterial Class II ketol‐acid reductoisomerase: Domain conservation and evolution

Publisher: Wiley

Date: 12-2005

DOI: 10.1110/PS.051791305

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.

Crystal structures of free, IMP-, and GMP-bound Escherichia coli hypoxanthine phosphoribosyltransferase

Publisher: Wiley

Date: 07-2002

DOI: 10.1110/PS.0201002

Abstract: Crystal structures have been determined for free Escherichia coli hypoxanthine phosphoribosyltransferase (HPRT) (2.9 A resolution) and for the enzyme in complex with the reaction products, inosine 5'-monophosphate (IMP) and guanosine 5'-monophosphate (GMP) (2.8 A resolution). Of the known 6-oxopurine phosphoribosyltransferase (PRTase) structures, E. coli HPRT is most similar in structure to that of Tritrichomonas foetus HGXPRT, with a rmsd for 150 Calpha atoms of 1.0 A. Comparison of the free and product bound structures shows that the side chain of Phe156 and the polypeptide backbone in this vicinity move to bind IMP or GMP. A nonproline cis peptide bond, also found in some other 6-oxopurine PRTases, is observed between Leu46 and Arg47 in both the free and complexed structures. For catalysis to occur, the 6-oxopurine PRTases have a requirement for alent metal ion, usually Mg(2+) in vivo. In the free structure, a Mg(2+) is coordinated to the side chains of Glu103 and Asp104. This interaction may be important for stabilization of the enzyme before catalysis. E. coli HPRT is unique among the known 6-oxopurine PRTases in that it exhibits a marked preference for hypoxanthine as substrate over both xanthine and guanine. The structures suggest that its substrate specificity is due to the modes of binding of the bases. In E. coli HPRT, the carbonyl oxygen of Asp163 would likely form a hydrogen bond with the 2-exocyclic nitrogen of guanine (in the HPRT-guanine-PRib-PP-Mg(2+) complex). However, hypoxanthine does not have a 2-exocyclic atom and the HPRT-IMP structure suggests that hypoxanthine is likely to occupy a different position in the purine-binding pocket.

Facile crystallization ofEscherichia coliketol-acid reductoisomerase

Publisher: International Union of Crystallography (IUCr)

Date: 21-07-2004

DOI: 10.1107/S0907444904012247

The structure of Human Microplasmin in Complex with Textilinin-1, an Aprotinin-like Inhibitor from the Australian Brown Snake

Publisher: Public Library of Science (PLoS)

Date: 15-01-2013

DOI: 10.1371/JOURNAL.PONE.0054104

Crystallization of the catalytic subunit ofSaccharomyces cerevisiaeacetohydroxyacid synthase

Publisher: International Union of Crystallography (IUCr)

Date: 23-08-2001

DOI: 10.1107/S0907444901011635

Abstract: Acetohydroxyacid synthase (AHAS E.C. 4.1.3.18) is the first enzyme in the biosynthetic pathway of the branched-chain amino acids isoleucine, leucine and valine. It is a thiamin diphosphate-dependent enzyme which catalyses the decarboxylation of pyruvate and its condensation with either 2-ketobutyrate or a second molecule of pyruvate to give 2-aceto-2-hydroxybutyrate or 2-acetolactate, respectively. The enzyme is the target of sulfonylurea and imidazolinone herbicides, which act as potent and specific inhibitors. Here, the crystallization and preliminary X-ray diffraction analysis of the catalytic subunit of Saccharomyces cerevisiae AHAS is reported. Data to 2.7 A resolution have been collected using synchrotron radiation (Advanced Photon Source, Chicago). Crystals have unit-cell parameters a = 95.8, b = 110.0, c = 178.9 A and belong to the space group P2(1)2(1)2(1). Preliminary analysis indicates there is one dimer located in each asymmetric unit.

Substrate-Promoted Formation of a Catalytically Competent Binuclear Center and Regulation of Reactivity in a Glycerophosphodiesterase from Enterobacter aerogenes

Publisher: American Chemical Society (ACS)

Date: 03-10-2008

DOI: 10.1021/JA803346W

Structural basis for the broad substrate specificity of two acyl-CoA dehydrogenases FadE5 from mycobacteria

Publisher: Proceedings of the National Academy of Sciences

Date: 29-06-2020

DOI: 10.1073/PNAS.2002835117

Abstract: The lipid content accounts for approximately 60% of the dry weight of the cell wall of pathogenic mycobacteria with Mycobacterium tuberculosis having more than 250 genes involved in fatty acid metabolism. Previous studies have shown that acyl-CoA dehydrogenase (ACD), which introduces unsaturation into fatty acids, exhibits strict substrate specificity toward different CoA thioester groups. Here, we identified a unique ACD member in mycobacteria that exhibits broad substrate specificity. Furthermore, we determined crystal structures of the enzyme and enzyme–substrate complexes to explain the broad substrate recognition observed in this system. Given the importance of FadE5 in fatty acid metabolism, these new structures are excellent platforms for rational structure based antituberculosis drug discovery.

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.

Crystallization ofArabidopsis thalianaacetohydroxyacid synthase in complex with the sulfonylurea herbicide chlorimuron ethyl

Publisher: International Union of Crystallography (IUCr)

Date: 18-12-2003

DOI: 10.1107/S0907444903025423

Abstract: Acetohydroxyacid synthase (AHAS EC 2.2.1.6) catalyses the formation of 2-acetolactate and 2-aceto-2-hydroxybutyrate as the first step in the biosynthesis of the branched-chain amino acids valine, leucine and isoleucine. The enzyme is inhibited by a wide range of substituted sulfonylureas and imidazolinones and many of these compounds are used as commercial herbicides. Here, the crystallization and preliminary X-ray diffraction analysis of the catalytic subunit of Arabidopsis thaliana AHAS in complex with the sulfonylurea herbicide chlorimuron ethyl are reported. This is the first report of the structure of any plant protein in complex with a commercial herbicide. Crystals diffract to 3.0 A resolution, have unit-cell parameters a = b = 179.92, c = 185.82 A and belong to space group P6(4)22. Preliminary analysis indicates that there is one monomer in the asymmetric unit and that these are arranged as pairs of dimers in the crystal. The dimers form a very open hexagonal lattice, with a high solvent content of 81%.

Author Correction: Cryo-EM structure of trimeric Mycobacterium smegmatis succinate dehydrogenase with a membrane-anchor SdhF

Publisher: Springer Science and Business Media LLC

Date: 23-02-2021

DOI: 10.1038/S41467-021-21616-3

Abstract: A Correction to this paper has been published: 0.1038/s41467-021-21616-3

Synthesis of purine N9-[2-hydroxy-3-O-(phosphonomethoxy)propyl] derivatives and their side-chain modified analogs as potential antimalarial agents

Publisher: Elsevier BV

Date: 02-2012

DOI: 10.1016/J.BMC.2011.12.034

Abstract: 6-Oxopurine acyclic nucleoside phosphonates (ANPs) have been shown to be potent inhibitors of hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT), a key enzyme of the purine salvage pathway in human malarial parasites. These compounds also exhibit antimalarial activity against parasites grown in culture. Here, a new series of ANPs, hypoxanthine and guanine 9-[2-hydroxy-3-(phosphonomethoxy)propyl] derivatives with different chemical substitutions in the 2'-position of the aliphatic chain were prepared and tested as inhibitors of Plasmodium falciparum (Pf) HGXPRT, Plasmodium vivax (Pv) HGPRT and human HGPRT. The attachment of an hydroxyl group to this position and the movement of the oxygen by one atom distal from N(9) in the purine ring compared with 2-(phosphonoethoxy)ethyl hypoxanthine (PEEHx) and 2-(phosphonoethoxy)ethyl guanine (PEEG) changes the affinity and selectivity for human HGPRT, PfHGXPRT and PvHGPRT. This is attributed to the differences in the three-dimensional structure of these inhibitors which affects their mode of binding. A novel observation is that these molecules are not always strictly competitive with 5-phospho-α-d-ribosyl-1-pyrophosphate. 9-[2-Hydroxy-3-(phosphonomethoxy)propyl]hypoxanthine (iso-HPMP-Hx) is a very weak inhibitor of human HGPRT but remains a good inhibitor of both the parasite enzymes with K(i) values of 2μM and 5μM for PfHGXPRT and PvHGPRT, respectively. The addition of pyrophosphate to the assay decreased the K(i) values for the parasite enzymes by sixfold. This suggests that the covalent attachment of a second group to the ANPs mimicking pyrophosphate and occupying its binding pocket could increase the affinity for these enzymes.

The crystal structure of Ba3V4O13

Publisher: Elsevier BV

Date: 12-1987

DOI: 10.1016/0022-4596(87)90247-7

A focused sulfated glycoconjugate Ugi library for probing heparan sulfate-binding angiogenic growth factors

Publisher: Elsevier BV

Date: 10-2012

DOI: 10.1016/J.BMCL.2012.08.001

Abstract: A library of small molecule heparan sulfate (HS) mimetics was synthesized by employing the Ugi four-component condensation of d-mannopyranoside-derived isocyanides with formaldehyde as the carbonyl component and a selection of carboxylic acids and amines, followed by sulfonation. The library was used to probe the subtle differences surrounding the ionic binding sites of three HS-binding angiogenic growth factors (FGF-1, FGF-2 and VEGF). Each compound features 3 or 4 sulfo groups which serve to anchor the ligand to the HS-binding site of the protein, with a erse array of functionality in place extending from C-1 or C-6 to probe for adjacent favorable binding interactions. Selectivity of binding to these proteins was clearly observed and supported by molecular docking calculations.

Composition differences between organic and conventional meat: a systematic literature review and meta-analysis

Publisher: Cambridge University Press (CUP)

Date: 16-02-2016

DOI: 10.1017/S0007114515005073

Abstract: Demand for organic meat is partially driven by consumer perceptions that organic foods are more nutritious than non-organic foods. However, there have been no systematic reviews comparing specifically the nutrient content of organic and conventionally produced meat. In this study, we report results of a meta-analysis based on sixty-seven published studies comparing the composition of organic and non-organic meat products. For many nutritionally relevant compounds (e.g. minerals, antioxidants and most in idual fatty acids (FA)), the evidence base was too weak for meaningful meta-analyses. However, significant differences in FA profiles were detected when data from all livestock species were pooled. Concentrations of SFA and MUFA were similar or slightly lower, respectively, in organic compared with conventional meat. Larger differences were detected for total PUFA and n -3 PUFA, which were an estimated 23 (95 % CI 11, 35) % and 47 (95 % CI 10, 84) % higher in organic meat, respectively. However, for these and many other composition parameters, for which meta-analyses found significant differences, heterogeneity was high, and this could be explained by differences between animal species/meat types. Evidence from controlled experimental studies indicates that the high grazing/forage-based diets prescribed under organic farming standards may be the main reason for differences in FA profiles. Further studies are required to enable meta-analyses for a wider range of parameters (e.g. antioxidant, vitamin and mineral concentrations) and to improve both precision and consistency of results for FA profiles for all species. Potential impacts of composition differences on human health are discussed.

Cryo-EM structure of trimeric Mycobacterium smegmatis succinate dehydrogenase with a membrane-anchor SdhF

Publisher: Springer Science and Business Media LLC

Date: 25-08-2020

DOI: 10.1038/S41467-020-18011-9

Abstract: Diheme-containing succinate:menaquinone oxidoreductases (Sdh) are widespread in Gram-positive bacteria but little is known about the catalytic mechanisms they employ for succinate oxidation by menaquinone. Here, we present the 2.8 Å cryo-electron microscopy structure of a Mycobacterium smegmatis Sdh, which forms a trimer. We identified the membrane-anchored SdhF as a subunit of the complex. The 3 kDa SdhF forms a single transmembrane helix and this helix plays a role in blocking the canonically proximal quinone-binding site. We also identified two distal quinone-binding sites with bound quinones. One distal binding site is formed by neighboring subunits of the complex. Our structure further reveals the electron roton transfer pathway for succinate oxidation by menaquinone. Moreover, this study provides further structural insights into the physiological significance of a trimeric respiratory complex II. The structure of the menaquinone binding site could provide a framework for the development of Sdh-selective anti-mycobacterial drugs.

Helicobacter pylori Xanthine–Guanine–Hypoxanthine PhosphoribosyltransferaseA Putative Target for Drug Discovery against Gastrointestinal Tract Infections

Publisher: American Chemical Society (ACS)

Date: 23-04-2021

DOI: 10.1021/ACS.JMEDCHEM.0C02184

The organophosphate-degrading enzyme from Agrobacterium radiobacter displays mechanistic flexibility for catalysis

Publisher: Portland Press Ltd.

Date: 25-11-2010

DOI: 10.1042/BJ20101054

Abstract: The OP (organophosphate)-degrading enzyme from Agrobacterium radiobacter (OpdA) is a binuclear metallohydrolase able to degrade highly toxic OP pesticides and nerve agents into less or non-toxic compounds. In the present study, the effect of metal ion substitutions and site-directed mutations on the catalytic properties of OpdA are investigated. The study shows the importance of both the metal ion composition and a hydrogen-bond network that connects the metal ion centre with the substrate-binding pocket using residues Arg254 and Tyr257 in the mechanism and substrate specificity of this enzyme. For the Co(II) derivative of OpdA two protonation equilibria (pKa1 ~5 pKa2 ~10) have been identified as relevant for catalysis, and a terminal hydroxide acts as the likely hydrolysis-initiating nucleophile. In contrast, the Zn(II) and Cd(II) derivatives only have one relevant protonation equilibrium (pKa ~4–5), and the μOH is the proposed nucleophile. The observed mechanistic flexibility may reconcile contrasting reaction models that have been published previously and may be beneficial for the rapid adaptation of OP-degrading enzymes to changing environmental pressures.

Structural basis for replicase polyprotein cleavage and substrate specificity of main protease from SARS-CoV-2

Publisher: Proceedings of the National Academy of Sciences

Date: 05-04-2022

DOI: 10.1073/PNAS.2117142119

Abstract: COVID-19 is a deadly r aging infectious disease with over 480 million cases worldwide. Unfortunately, effective therapies remain very limited. Novel antiviral agents are urgently needed to combat this global healthcare crisis. Here, we elucidate the structural basis for replicase polyprotein cleavage and substrate specificity of SARS-CoV-2 main protease (M pro ). Through analyzing a series of high-resolution structures of SARS-CoV-2 M pro throughout the proteolytic process, we demonstrate the molecular mechanism of M pro in proteolytic processing that confers substrate specificity. Substrate selectivity is revealed using structures of the H41A mutant in complex with six in idual native cleavage substrates. Our study underscores the mechanistic function of M pro in the viral life cycle, which provides structural insights to develop effective inhibitors against this essential target of SARS-CoV-2.

Three-Dimensional Structure of the α-Conotoxin GI at 1.2 Å Resolution^,

Publisher: American Chemical Society (ACS)

Date: 1996

DOI: 10.1021/BI960820H

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

Structure of the RNA-dependent RNA polymerase from COVID-19 virus

Publisher: American Association for the Advancement of Science (AAAS)

Date: 15-05-2202

DOI: 10.1126/SCIENCE.ABB7498

Abstract: Many in the scientific community have mobilized to understand the virus that is causing the global coronavirus disease 2019 (COVID-19) pandemic. Gao et al. focused on a complex that plays a key role in the replication and transcription cycle of the virus. They used cryo–electron microscopy to determine a 2.9-angstrom-resolution structure of the RNA-dependent RNA polymerase nsp12, which catalyzes the synthesis of viral RNA, in complex with two cofactors, nsp7 and nsp8. nsp12 is a target for nucleotide analog antiviral inhibitors such as remdesivir, and the structure may provide a basis for designing new antiviral therapeutics. Science , this issue p. 779

Crystallization and preliminary crystallographic data of the fab fragment of an anti-phenylalanine hydroxylase monoclonal antibody

Publisher: Elsevier BV

Date: 11-1985

DOI: 10.1016/0022-2836(85)90121-4

Abstract: Two crystal habits, one rod shaped and the other square prismatic, of the Fab fragment of a monoclonal anti-phenylalanine hydroxylase antibody have been grown using the method of vapour phase diffusion against polyethylene glycol 6000. The square prisms diffract to better than 2.8 A, belong to the space group P1 and have unit cell parameters a = 41.8 A, b = 50.3 A, c = 114.7 A, alpha = 97.6 degrees, beta = 91.7 degrees, gamma = 91.0 degrees, while the rod-shaped crystals belong to the space group P212121, have unit cell parameters a = 105.6 A, b = 119.8 A, c = 82.2 A and diffract to 3.5 A resolution.

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.

Structure-activity relationships of bensulfuron methyl and its derivatives as novel agents against drug-resistant Candida auris

Publisher: Wiley

Date: 08-10-2023

DOI: 10.1111/CBDD.14364

The structure–activity relationship in herbicidal monosubstituted sulfonylureas

Publisher: Wiley

Date: 02-11-2011

DOI: 10.1002/PS.2305

Abstract: The herbicide sulfonylurea (SU) belongs to one of the most important class of herbicides worldwide. It is well known for its ecofriendly, extreme low toxicity towards mammals and ultralow dosage application. The original inventor, G Levitt, set out structure-activity relationship (SAR) guidelines for SU structural design to attain superhigh bioactivity. A new approach to SU molecular design has been developed. After the analysis of scores of SU products by X-ray diffraction methodology and after greenhouse herbicidal screening of 900 novel SU structures synthesised in the authors' laboratory, it was found that several SU structures containing a monosubstituted pyrimidine moiety retain excellent herbicidal characteristics, which has led to partial revision of the Levitt guidelines. Among the novel SU molecules, monosulfuron and monosulfuron-ester have been developed into two new herbicides that have been officially approved for field application and applied in millet and wheat fields in China. A systematic structural study of the new substrate-target complex and the relative mode of action in comparison with conventional SU has been carried out. A new mode of action has been postulated.

Crystallization of an Fv fragment from a human IgM cryoglobulin by a microseeding technique

Publisher: Elsevier BV

Date: 1993

DOI: 10.1016/0022-0248(93)90030-Z

Crystal structure of textilinin-1, a Kunitz-type serine protease inhibitor from the venom of the Australian common brown snake (Pseudonaja textilis)

Publisher: Wiley

Date: 08-05-2009

DOI: 10.1111/J.1742-4658.2009.07034.X

Abstract: Textilinin-1 is a Kunitz-type serine protease inhibitor isolated from the venom of the Australian common brown snake, Pseudonaja textilis. This molecule binds to and blocks the activity of a range of serine proteases, including plasmin and trypsin. Textilinin-1's ability to inhibit plasmin, a protease involved in fibrinolysis, has raised the possibility that it could be used as an alternative to aprotinin (Trasylol) as a systemic antibleeding agent in surgery. Here, the crystal structure of free recombinant textilinin-1 has been determined to 1.63 A, with three molecules observed in the asymmetric unit. All of these have a similar overall fold to aprotinin, except that the canonical loop for one of the molecules is inverted such that the side chain of the P1' residue, Val18, is partially buried by intramolecular contacts to Pro15, Thr13, and Ile36. In aprotinin, the P1' residue is Ala16, whose side chain is too small to form similar contacts. The loop inversion in textilinin-1 is facilitated by changes in backbone dihedral angles for the P1 and P2' residues, such that they alternate between values in the beta-sheet and alpha-helical regions of the Ramachandran plot. In a comparison with the structures of all other known Kunitz-type serine protease inhibitors, no such conformational variability has been observed. The presence of the bulkier valine as the P1' residue in textilinin-1 appears to be a major contributor to reducing the binding affinity for plasmin as compared to aprotinin (3.5 nm versus 0.053 nm) and could also account for an observed narrower binding specificity.

Sulfide, sulfoxide and sulfone bridged acyclic nucleoside phosphonates as inhibitors of the Plasmodium falciparum and human 6-oxopurine phosphoribosyltransferases: Synthesis and evaluation

Publisher: Elsevier BV

Date: 12-2019

DOI: 10.1016/J.EJMECH.2019.111667

Abstract: Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) is a recognized target for antimalarial chemotherapeutics. It synthesises all of the 6-oxopurine nucleoside monophosphates, IMP, GMP and XMP needed by the malarial parasite, Plasmodium falciparum (Pf). PfHGXPRT is also indirectly responsible for the synthesis of the adenosine monophosphate, AMP. The acyclic nucleoside phosphonates (ANPs) are a class of PfHGXPRT inhibitors. Prodrugs of these compounds are able to arrest the growth of Pf in cell culture. In the search for new inhibitors of PfHGXPRT, a series of sulfur containing ANPs (thia-ANPs) has been designed and synthesized. These compounds are based on the structure of 2-(phosphonoethoxy)ethylguanine (PEEG) and PEEHx which consist of a purine base (i.e. guanine or hypoxanthine) linked to a phosphonate group by five atoms i.e. four carbons and one oxygen. Here, PEEG and PEEHx were modified by substituting a sulfide, sulfoxide or a sulfone bridge for the oxygen atom in the linker. The effect of these substitutions on the K

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

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.

Molecular Basis of Sulfonylurea Herbicide Inhibition of Acetohydroxyacid Synthase

Publisher: Elsevier BV

Date: 02-2003

DOI: 10.1074/JBC.M211648200

Systematic characterization of mutations in yeast acetohydroxyacid synthase

Publisher: Wiley

Date: 12-06-2003

DOI: 10.1046/J.1432-1033.2003.03671.X

Abstract: Acetohydroxyacid synthase (AHAS, EC 4.1.3.18) catalyses the first step in branched-chain amino acid biosynthesis and is the target for sulfonylurea and imidazolinone herbicides, which act as potent and specific inhibitors. Mutants of the enzyme have been identified that are resistant to particular herbicides. However, the selectivity of these mutants towards various sulfonylureas and imidazolinones has not been determined systematically. Now that the structure of the yeast enzyme is known, both in the absence and presence of a bound herbicide, a detailed understanding of the molecular interactions between the enzyme and its inhibitors becomes possible. Here we construct 10 active mutants of yeast AHAS, purify the enzymes and determine their sensitivity to six sulfonylureas and three imidazolinones. An additional three active mutants were constructed with a view to increasing imidazolinone sensitivity. These three variants were purified and tested for their sensitivity to the imidazolinones only. Substantial differences are observed in the sensitivity of the 13 mutants to the various inhibitors and these differences are interpreted in terms of the structure of the herbicide-binding site on the enzyme.

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

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

Plasmodium vivax hypoxanthine-guanine phosphoribosyltransferase: A target for anti-malarial chemotherapy

Publisher: Elsevier BV

Date: 10-2010

DOI: 10.1016/J.MOLBIOPARA.2010.05.018

Abstract: The malarial parasite, Plasmodium vivax (Pv), causes a serious infectious disease found primarily in Asia and the Americas. For protozoan parasites, 6-oxopurine phosphoribosyltransferases (PRTases) provide the only metabolic pathway to synthesize the purine nucleoside monophosphates essential for DNA/RNA production. We have purified the recombinant Pv 6-oxopurine (PRTase) and compared its properties with the human and Pf enzymes. The Pv enzyme uses hypoxanthine and guanine with similar catalytic efficiency to the Pf enzyme but xanthine is not a substrate, hence we identify this enzyme as PvHGPRT. Mass spectrometry suggests that PvHGPRT contains bound magnesium ions that are removed by EDTA resulting in loss of activity. However, the addition of Mg(2+) restores activity. Acyclic nucleoside phosphonates (ANPs) are good inhibitors of PvHGPRT having K(i) values as low as 3 microM. These compounds can form the basis for the design of new drugs aimed at combating malaria caused by Pv.

Difunctionalization of primary aromatic amines with ethylene; synthesis of μ-arylnitrilobis(ethane-2,1-diyl)dimercury(II) complexes. Crystal structure of 2,4,6-Me3C6H2N-(CH2CH2HgCl)2

Publisher: Elsevier BV

Date: 10-1989

DOI: 10.1016/0022-328X(89)85095-8

Lead compounds for antimalarial chemotherapy: purine base analogs discriminate between human and P. falciparum 6-oxopurine phosphoribosyltransferases.

Publisher: American Chemical Society (ACS)

Date: 16-11-2006

DOI: 10.1021/JM061012J

Abstract: The malarial parasite Plasmodium falciparum depends on the purine salvage enzyme hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) to convert purine bases from the host to nucleotides needed for DNA and RNA synthesis. An approach to developing antimalarial drugs is to use HGXPRT to convert introduced purine base analogs to nucleotides that are toxic to the parasite. This strategy requires that these compounds be good substrates for the parasite enzyme but poor substrates for the human counterpart, HGPRT. Bases with a chlorine atom in the 6-position or a nitrogen in the 8-position exhibited strong discrimination between P. falciparum HGXPRT and human HGPRT. The k(cat)/K(m) values for the Plasmodium enzyme using 6-chloroguanine and 8-azaguanine as substrates were 50 - 80-fold and 336-fold higher than for the human enzyme, respectively. These and other bases were effective in inhibiting the growth of the parasite in vitro, giving IC(50) values as low as 1 microM.

The Crystal Structure of Free Human Hypoxanthine-guanine Phosphoribosyltransferase Reveals Extensive Conformational Plasticity Throughout the Catalytic Cycle

Publisher: Elsevier BV

Date: 08-2005

DOI: 10.1016/J.JMB.2005.05.061

Abstract: Human hypoxanthine-guanine phosphoribosyltransferase (HGPRT) catalyses the synthesis of the purine nucleoside monophosphates, IMP and GMP, by the addition of a 6-oxopurine base, either hypoxanthine or guanine, to the 1-beta-position of 5-phospho-alpha-d-ribosyl-1-pyrophosphate (PRib-PP). The mechanism is sequential, with PRib-PP binding to the free enzyme prior to the base. After the covalent reaction, pyrophosphate is released followed by the nucleoside monophosphate. A number of snapshots of the structure of this enzyme along the reaction pathway have been captured. These include the structure in the presence of the inactive purine base analogue, 7-hydroxy [4,3-d] pyrazolo pyrimidine (HPP) and PRib-PP.Mg2+, and in complex with IMP or GMP. The third structure is that of the immucillinHP.Mg(2+).PP(i) complex, a transition-state analogue. Here, the first crystal structure of free human HGPRT is reported to 1.9A resolution, showing that significant conformational changes have to occur for the substrate(s) to bind and for catalysis to proceed. Included in these changes are relative movement of subunits within the tetramer, rotation and extension of an active-site alpha-helix (D137-D153), reorientation of key active-site residues K68, D137 and K165, and the rearrangement of three active-site loops (100-128, 165-173 and 186-196). Toxoplasma gondii HGXPRT is the only other 6-oxopurine phosphoribosyltransferase structure solved in the absence of ligands. Comparison of this structure with human HGPRT reveals significant differences in the two active sites, including the structure of the flexible loop containing K68 (human) or K79 (T.gondii).

Acetohydroxyacid Synthase: A Target for Antimicrobial Drug Discovery

Publisher: Bentham Science Publishers Ltd.

Date: 02-2014

DOI: 10.2174/13816128113199990009

Abstract: Acetohydroxyacid synthase (AHAS) (EC 2.2.1.6) (also known as acetolactate synthase) is the first common enzyme in the branched chain amino acid (BCAA) biosynthesis pathway. This pathway is present in microorganisms and in plants but not in animals, making it an attractive target for both drug and herbicide discovery. The function of AHAS is to catalyze the conversion of two molecules of pyruvate to 2-acetolactate or to convert one molecule of pyruvate and a molecule of 2-ketobutyrate into 2-aceto-2-hydroxybutyrate. Three cofactors are required for the activity of AHAS: thiamine diphosphate (ThDP), Mg²⁺ and flavin-adenine dinucleotide (FAD). AHAS is the target for several classes of commercial herbicides that include the sulfonylurea and imidazolinone families. These herbicides are potent and selective inhibitors of AHAS with Ki values that can be in the low nM range. Such compounds also exhibit low application rates as herbicides (typically ~3 g ha⁻¹) and have low mammalian toxicity (LD₅₀ values typically >4g/kg), thereby highlighting their utility and effectiveness as biocidal agents. However, somewhat surprisingly given the central importance of AHAS in the metabolism of microorganisms, no inhibitors of this enzyme have been commercialized into antimicrobial agents. Here we provide an overview of the biochemical characterization of AHASs from bacterial and fungal sources, analyse the structural features of these enzymes that are criticial to catalysis andprovide the current data on AHAS inhibitors that have potential to be developed into antimicrobial therapeutics.

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.

Molecular recognition of trehalose and trehalose analogues by Mycobacterium tuberculosis LpqY-SugABC

Publisher: Proceedings of the National Academy of Sciences

Date: 21-08-2023

DOI: 10.1073/PNAS.2307625120

Abstract: Trehalose plays a crucial role in the survival and virulence of the deadly human pathogen Mycobacterium tuberculosis ( Mtb ). The type I ATP-binding cassette (ABC) transporter LpqY-SugABC is the sole pathway for trehalose to enter Mtb . The substrate-binding protein, LpqY, which forms a stable complex with the translocator SugABC, recognizes and captures trehalose and its analogues in the periplasmic space, but the precise molecular mechanism for this process is still not well understood. This study reports a 3.02-Å cryoelectron microscopy structure of trehalose-bound Mtb LpqY-SugABC in the pretranslocation state, a crystal structure of Mtb LpqY in a closed form with trehalose bound and five crystal structures of Mtb LpqY in complex with different trehalose analogues. These structures, accompanied by substrate-stimulated ATPase activity data, reveal how LpqY recognizes and binds trehalose and its analogues, and highlight the flexibility in the substrate binding pocket of LpqY. These data provide critical insights into the design of trehalose analogues that could serve as potential molecular probe tools or as anti-TB drugs.

Plant collagenase: Unique collagenolytic activity of cysteine proteases from ginger

Publisher: Elsevier BV

Date: 12-2007

DOI: 10.1016/J.BBAGEN.2007.08.003

Abstract: Two cysteine proteases, GP2 and GP3, have been isolated from ginger rhizomes (Zingiber officinale). GP2 is virtually identical to a previously identified ginger protease GPII [K.H. Choi, and R.A. Laursen, Amino-acid sequence and glycan structures of cysteine proteases with proline specificity from ginger rhizome Zingiber officinale, Eur. J. Biochem. 267 (2000) 1516-1526.], and cleaves native type I collagen at multiple discrete sites, which are in the interior of the triple helical region of this molecule. In reaction with proline-containing peptides GP2 shows preference for Pro in the P2 position, and at least 10-fold higher efficiency of hydrolysis than papain. Comparison of models of GP2 and GP3 with the crystal structure of papain shows that the three enzymes have different S2 pocket structures. The S2 pocket in GP2 and GP3 is half the size of that of papain. GP2 is the only reported plant cysteine protease with a demonstrated ability to hydrolyse native collagen. The results support a role for ginger proteases as an alternative to papain, in commercial applications such as meat tenderization, where collagen is the target substrate.

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

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

The Crystal Structures of Klebsiella pneumoniae Acetolactate Synthase with Enzyme-bound Cofactor and with an Unusual Intermediate

Publisher: Elsevier BV

Date: 2004

DOI: 10.1074/JBC.M304038200

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.

Herbicides That Target Acetohydroxyacid Synthase Are Potent Inhibitors of the Growth of Drug-Resistant Candida auris

Publisher: American Chemical Society (ACS)

Date: 28-09-2020

DOI: 10.1021/ACSINFECDIS.0C00229

Structure and mechanism of inhibition of plant acetohydroxyacid synthase

Publisher: Elsevier BV

Date: 03-2008

DOI: 10.1016/J.PLAPHY.2007.12.004

Abstract: Plants and microorganisms synthesize valine, leucine and isoleucine via a common pathway in which the first reaction is catalysed by acetohydroxyacid synthase (AHAS, EC 2.2.1.6). This enzyme is of substantial importance because it is the target of several herbicides, including all members of the popular sulfonylurea and imidazolinone families. However, the emergence of resistant weeds due to mutations that interfere with the inhibition of AHAS is now a worldwide problem. Here we summarize recent ideas on the way in which these herbicides inhibit the enzyme, based on the 3D structure of Arabidopsis thaliana AHAS. This structure also reveals important clues for understanding how various mutations can lead to herbicide resistance.

Aspartic Proteases ofPlasmodium falciparumas the Target of HIV‐1 Protease Inhibitors

Publisher: Oxford University Press (OUP)

Date: 15-04-2005

DOI: 10.1086/428781

“It takes a whole school to raise a teacher”: examining executive staff support and perception of casual relief teachers in Australian schools

Publisher: Springer Science and Business Media LLC

Date: 09-06-2021

DOI: 10.1007/S13384-021-00445-9

Design of Plasmodium vivax Hypoxanthine-Guanine Phosphoribosyltransferase Inhibitors as Potential Antimalarial Therapeutics.

Publisher: American Chemical Society (ACS)

Date: 05-12-2018

DOI: 10.1021/ACSCHEMBIO.7B00916

Abstract: Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) are the foremost causative agents of malaria. Due to the development of resistance to current antimalarial medications, new drugs for this parasitic disease need to be discovered. The activity of hypoxanthine-guanine-[xanthine]-phosphoribosyltransferase, HG[X]PRT, is reported to be essential for the growth of both of these parasites, making it an excellent target for antimalarial drug discovery. Here, we have used rational structure-based methods to design an inhibitor, [3R,4R]-4-guanin-9-yl-3-((S)-2-hydroxy-2-phosphonoethyl)oxy-1-N-(phosphonopropionyl)pyrrolidine, of PvHGPRT and PfHGXPRT that has K

Crystallization and preliminary X-ray diffraction studies of mammalian purple acid phosphatase

Publisher: International Union of Crystallography (IUCr)

Date: 08-1999

DOI: 10.1107/S0907444999006241

Abstract: The oxidized form of purple acid phosphatase from pig allantoic fluid has been crystallized in the presence of phosphate using the hanging-drop technique. The crystals belong to the space group P 2 1 2 1 2 1 and have unit-cell parameters a = 66.8, b = 70.3, c = 78.7 Å. Diffraction data collected from a cryocooled crystal using a conventional X-ray source extend to 1.55 Å resolution. A knowledge of the three-dimensional structure of mammalian purple acid phosphatase will aid in understanding the substrate specificity of the enzyme and will be important in the rational design of inhibitors, with potential in the treatment of bone diseases.

6-Oxopurine Phosphoribosyltransferase: A Target for the Development of Antimalarial Drugs

Publisher: Bentham Science Publishers Ltd.

Date: 08-2011

DOI: 10.2174/156802611796575911

Abstract: Malaria remains the most serious parasitic diseases affecting humans in the world today, resulting in 1-2 million fatalities each year. Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) are the predominant causative agents. Both are responsible for widespread mortality and morbidity and are a serious socio-economic burden, especially for countries in the developing world. One of the most important defences against malaria has been the use of chemotherapeutic drugs (e.g. chloroquine, artemisinins, pyrimethamine) but these have mainly been found by serendipity. Their mechanisms was not understood at the time of their discovery and, even today, are still not unequivocal. For many of these compounds, the parasite is now resistant and, hence, there is an urgent need to develop new therapeutic drugs directed to validated targets. One metabolic pathway crucial for the survival and replication and survival of the parasite is the synthesis of the purine nucleoside monophosphates essential for the production of DNA/RNA molecules. A key enzyme in this pathway is the 6-oxopurine phosphoribosyltransferase (PRTase). The focus of this review is on the identification and characterization of inhibitors of the enzymes from both Pf and Pv as antimalarial drug leads. The acyclic nucleoside phosphonates (ANPs) appear to be excellent candidates because they are good inhibitors of the two Plasmodium enzymes, can be selective compared to the human enzyme, can arrest parasitemia in cell based assays, have low cytotoxicity to the human host cell and, because of their stable carbon-phosphorous bond, are stable within the cell.

Structural basis of trehalose recycling by the ABC transporter LpqY-SugABC

Publisher: American Association for the Advancement of Science (AAAS)

Date: 30-10-2020

DOI: 10.1126/SCIADV.ABB9833

Abstract: Four cryo-EM structures of LpqY-SugABC complex reveal the mechanism of trehalose transport across the membrane to cytoplasm.

The development of teacher self-efficacy from preservice to early career teacher: a systematic review of development and methodological quality in longitudinal research

Publisher: Informa UK Limited

Date: 20-10-2022

DOI: 10.1080/1743727X.2021.1990879

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.

The effect of novel [3-fluoro-(2-phosphonoethoxy)propyl]purines on the inhibition of Plasmodium falciparum, Plasmodium vivax and human hypoxanthine–guanine–(xanthine) phosphoribosyltransferases

Publisher: Elsevier BV

Date: 09-2013

DOI: 10.1016/J.EJMECH.2013.06.032

Abstract: Protozoan parasites from the Plasmodiidae family are the causative agents of malaria. Inhibition of hypoxanthine-guanine-(xanthine) phosphoribosyltransferase (HG(X)PRT) has been suggested as a target for development of new anti-malarial therapeutics. Acyclic nucleoside phosphonates (ANPs) are potent and selective inhibitors of plasmodial HG(X)PRTs. A new series of ANPs, based on the chemical structure and inhibitory activity of three ANPs, 2-(phosphonoethoxy)ethyl with either guanine or hypoxanthine as the base (PEEG and PEEHx) and 3-hydroxy-2-(phosphonomethoxy)propyl with guanine as the base (HPMPG), were prepared. These compounds are stereoisomers of 3-fluoro-(2-phosphonoethoxy)propyl (FPEPs) and 3-fluoro-(2-phosphonomethoxy)propyl (FPMPs) analogues. Both the (R)- and (S)-isomers of these fluorinated derivatives have higher Ki values (by 10- to 1000-fold) for human HGPRT and Plasmodium falciparum HGXPRT than the non-fluorinated ANPs. Possible explanations for these changes in affinity are proposed based on docking studies using the known crystal structures of human HGPRT in complex with PEEG.

Editorial [Hot Topic:Drug Targets for the Treatment of Protozoan Parasitic Diseases (Guest Editor: Luke Guddat)]

Publisher: Bentham Science Publishers Ltd.

Date: 08-2011

DOI: 10.2174/156802611796575920

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 of Mycobacterium tuberculosis cytochrome bcc in complex with Q203 and TB47, two anti-TB drug candidates

Publisher: eLife Sciences Publications, Ltd

Date: 25-11-2021

DOI: 10.7554/ELIFE.69418

Abstract: Pathogenic mycobacteria pose a sustained threat to global human health. Recently, cytochrome bcc complexes have gained interest as targets for antibiotic drug development. However, there is currently no structural information for the cytochrome bcc complex from these pathogenic mycobacteria. Here, we report the structures of Mycobacterium tuberculosis cytochrome bcc alone (2.68 Å resolution) and in complex with clinical drug candidates Q203 (2.67 Å resolution) and TB47 (2.93 Å resolution) determined by single-particle cryo-electron microscopy. M. tuberculosis cytochrome bcc forms a dimeric assembly with endogenous menaquinone/menaquinol bound at the quinone/quinol-binding pockets. We observe Q203 and TB47 bound at the quinol-binding site and stabilized by hydrogen bonds with the side chains of QcrB Thr 313 and QcrB Glu 314 , residues that are conserved across pathogenic mycobacteria. These high-resolution images provide a basis for the design of new mycobacterial cytochrome bcc inhibitors that could be developed into broad-spectrum drugs to treat mycobacterial infections.

Cryo-EM structure of mycobacterial cytochrome bd reveals two oxygen access channels

Publisher: Springer Science and Business Media LLC

Date: 30-07-2021

DOI: 10.1038/S41467-021-24924-W

Abstract: Cytochromes bd are ubiquitous amongst prokaryotes including many human-pathogenic bacteria. Such complexes are targets for the development of antimicrobial drugs. However, an understanding of the relationship between the structure and functional mechanisms of these oxidases is incomplete. Here, we have determined the 2.8 Å structure of Mycobacterium smegmatis cytochrome bd by single-particle cryo-electron microscopy. This bd oxidase consists of two subunits CydA and CydB, that adopt a pseudo two-fold symmetrical arrangement. The structural topology of its Q-loop domain, whose function is to bind the substrate, quinol, is significantly different compared to the C-terminal region reported for cytochromes bd from Geobacillus thermodenitrificans ( G. th ) and Escherichia coli ( E. coli ). In addition, we have identified two potential oxygen access channels in the structure and shown that similar tunnels also exist in G. th and E. coli cytochromes bd . This study provides insights to develop a framework for the rational design of antituberculosis compounds that block the oxygen access channels of this oxidase.

Author response: Structure of Mycobacterium tuberculosis cytochrome bcc in complex with Q203 and TB47, two anti-TB drug candidates

Publisher: eLife Sciences Publications, Ltd

Date: 02-09-2021

DOI: 10.7554/ELIFE.69418.SA2

Synthesis of Novel N-Branched Acyclic Nucleoside Phosphonates As Potent and Selective Inhibitors of Human, Plasmodium falciparum and Plasmodium vivax 6-Oxopurine Phosphoribosyltransferases

Publisher: American Chemical Society (ACS)

Date: 22-06-2012

DOI: 10.1021/JM300662D

Abstract: Hypoxanthine-guanine-(xanthine) phosphoribosyltransferase (HG(X)PRT) is crucial for the survival of malarial parasites Plasmodium falciparum (Pf) and Plasmodium vivax (Pv). Acyclic nucleoside phosphonates (ANPs) are inhibitors of HG(X)PRT and arrest the growth of Pf in cell culture. Here, a novel class of ANPs containing trisubstituted nitrogen (aza-ANPs) has been synthesized. These compounds have a wide range of K(i) values and selectivity for human HGPRT, PfHGXPRT, and PvHGPRT. The most selective and potent inhibitor of PfHGXPRT is 9-[N-(3-methoxy-3-oxopropyl)-N-(2-phosphonoethyl)-2-aminoethyl]hypoxanthine (K(i) = 100 nM): no inhibition could be detected against the human enzyme. This compound exhibits the highest ever reported selectivity for PfHGXPRT compared to human HGPRT. For PvHGPRT, 9-[N-(2-carboxyethyl)-N-(2-phosphonoethyl)-2-aminoethyl]guanine has a K(i) of 50 nM, the best inhibitor discovered for this enzyme to date. Docking of these compounds into the known structures of human HGPRT in complex with ANP-based inhibitors suggests reasons for the variations in affinity, providing insights for the design of antimalarial drug candidates.

Conformational Changes in a Plant Ketol-Acid Reductoisomerase upon Mg2+ and NADPH Binding as Revealed by Two Crystal Structures

Publisher: Elsevier BV

Date: 05-2009

DOI: 10.1016/J.JMB.2009.04.012

Abstract: Ketol-acid reductoisomerase (KARI EC 1.1.1.86) is an enzyme in the branched-chain amino acid biosynthesis pathway where it catalyzes the conversion of 2-acetolactate into (2R)-2,3-dihydroxy-3-isovalerate or the conversion of 2-aceto-2-hydroxybutyrate into (2R,3R)-2,3-dihydroxy-3-methylvalerate. KARI catalyzes two reactions-alkyl migration and reduction-and requires Mg(2+) and NADPH for activity. To date, the only reported structures for a plant KARI are those of the spinach enzyme-Mn(2+)-(phospho)ADP ribose-(2R,3R)-2,3-dihydroxy-3-methylvalerate complex and the spinach KARI-Mg(2)(+)-NADPH-N-hydroxy-N-isopropyloxamate complex, where N-hydroxy-N-isopropyloxamate is a predicted transition-state analog. These studies demonstrated that the enzyme consists of two domains, N-domain and C-domain, with the active site at the interface of these domains. Here, we have determined the structures of the rice KARI-Mg(2+) and rice KARI-Mg(2)(+)-NADPH complexes to 1.55 A and 2.80 A resolutions, respectively. In comparing the structures of all the complexes, several differences are observed. Firstly, the N-domain is rotated up to 15 degrees relative to the C-domain, expanding the active site by up to 4 A. Secondly, an alpha-helix in the C-domain that includes residues V510-T519 and forms part of the active site moves by approximately 3.9 A upon binding of NADPH. Thirdly, the 15 C-terminal amino acid residues in the rice KARI-Mg(2+) complex are disordered. In the rice KARI-Mg(2)(+)-NADPH complex and the spinach KARI structures, many of the 15 residues bind to NADPH and the N-domain and cover the active site. Fourthly, the location of the metal ions within the active site can vary by up to 2.7 A. The new structures allow us to propose that an induced-fit mechanism operates to (i) allow substrate to enter the active site, (ii) close over the active site during catalysis, and (iii) open the active site to facilitate product release.

Three-dimensional structure of a human Fab with high affinity for tetanus toxoid

Publisher: Elsevier BV

Date: 1998

DOI: 10.1016/S1380-2933(97)10003-3

Abstract: The wide range of antibody specificity and affinity results from the differing shapes and chemical compositions of their binding sites. These shapes range from discrete grooves in antibodies elicited by linear oligomers of nucleotides and carbohydrates to shallow depressions or flat surfaces for accommodation of proteins, peptides and large organic compounds. To determine the Fab structure of a high-affinity human antitoxin antibody. To explore structural features which enable the antibody to bind to intact tetanus toxoid, peptides derived from the sequence of the natural immunogen and antigenic mimics identified by combinatorial chemistry. To explain why this Fab shows a remarkable tendency to produce crystals consistently diffracting to d spacings of 1.7-1.8 A. To use this information to engineer a strong tendency to crystallize into the design of other Fabs. The protein was crystallized in hanging or sitting drops by a microseeding technique in polyethylene glycol (PEG) 8000. Crystals were subjected to X-ray analysis and the three-dimensional structure of the Fab was determined by the molecular replacement method. Interactive computer graphics were employed to fit models to electron density maps, survey the structure in multiple views and discover the crystal packing motif of the protein. Exceptionally large single crystals of this protein have been obtained, one measuring 5 x 3 x 2 mm (l x w x d). The latter was cut into six irregular pieces, each retaining the features of the original in diffracting to high resolution (1.8 A) with little decay in the X-ray beam. In an in idual Fab, the active site is relatively flat and it seems likely that the protein antigen and derivative peptides are tightly held on the outer surface without significant penetration into the interior. There is no free space to accommodate even a dipeptide between VH and VL. One of the unique features of the B7-15A2 Fab is a large aliphatic ridge dominating the center of the active site. The CDR3 of the H chain contributes significantly to this ridge, as well as to adjoining regions projected to be important for the docking of the antigen. Both the ease of crystallization and the favorable diffraction properties are mainly attributable to the tight packing of the protein molecules in the crystal lattice. The B7-15A2 active site provides a stable and well defined platform for high affinity docking of proteins, peptides and their mimotopes. The advantages for future developments are suggested by the analysis of the crystal properties. It should be possible to incorporate the features promoting crystallization, close packing and resistance to radiation damage into engineered human antibodies without altering the desired specificities and affinities of their active sites.

Crystallographic Analysis of Antigen–Antibody Complexes: End-on Insertion of Ligands in Antibodies—CDR3 Loops as Arbiters

Publisher: Elsevier BV

Date: 06-1996

DOI: 10.1006/METH.1996.0062

Abstract: As the dominant constituents of the active sites, complementarity-determining regions (CDRs) and particularly the CDR3 loops strongly influence the size and shape of this interdomain space. Six sets of CDR3 loops were extracted from our collection of crystal structures and examined for their modes of association. The CDR3 loops of the NC6.8 Fab face each other across a small crevice that is expanded further by end-on insertion of its high-affinity ligand (a trisubstituted guanidine sweet-tasting compound). This wedging event triggers a series of extensive local and transmitted conformational changes. In the 4-4-20 Fab, the CDR3 loops provide scaffolding for the high-affinity binding of fluorescein and shield the ligand from bulk solvent in the interdomain space above and below the very compact binding slot. Constituents of the CDR2 and CDR3 loops of the BV04-01 Fab interact to form "false floors" over potential cavity-type sites and thereby eliminate end-on insertion. Instead, fragments of single-stranded DNA are bound with low affinity in a groove whose course is altered on complex formation by global movements of VH relative to VL and by local shifts of HCDR3. Trafficking of even small peptide ligands between the V domains of the Pot Fv is prevented by the collapse of the large HCDR3 segment into the residual interdomain space. This protein is better suited to polyreactive binding of a variety of large protein antigens on its external surfaces. By comparison, the space available between the CDR3 loops of the Mcg light-chain dimer is very large. It has proved to be accessible for end-on insertion of peptides and other ligands ranging over seven orders of magnitude in affinity. Recently, an insect neuropeptide hormone, with pGlu as its penetrating agent, has been found to pierce the entire V dimer interface from the entrance of the traditional active site to the solvent pool between the V and C domains. In an Mcg x Hud heterodimer, the Hud CDR3 plays a role similar to that of the Pot H chain and blocks access to the interdomain space by close interactions with the CDR3 of Mcg.

Crystal Structures of Intact IgG Antibodies

Publisher: Elsevier BV

Date: 12-1993

DOI: 10.1006/IMMU.1993.1054

Crystal structure of yeast acetohydroxyacid synthase: a target for herbicidal inhibitors

Publisher: Elsevier BV

Date: 03-2002

DOI: 10.1006/JMBI.2001.5419

The effect of fast protein liquid chromatography (FPLC) cationic exchange purification on the crystallization of a monoclonal Fab fragment

Publisher: Elsevier BV

Date: 05-1988

DOI: 10.1016/0003-2697(88)90649-5

Abstract: Fast protein liquid chromatography cationic exchange purification to homogeneity of a monoclonal Fab fragment has resulted in an improvement in the quality of crystals for X-ray diffraction studies. This improvement is displayed in a well-defined crystal morphology, reproducibility of crystal growth, and increased resolution of diffraction data.

Crystal structure of mammalian purple acid phosphatase

Publisher: Elsevier BV

Date: 07-1999

DOI: 10.1016/S0969-2126(99)80100-2

Abstract: Mammalian purple acid phosphatases are highly conserved binuclear metal-containing enzymes produced by osteoclasts, the cells that resorb bone. The enzyme is a target for drug design because there is strong evidence that it is involved in bone resorption. The 1.55 A resolution structure of pig purple acid phosphatase has been solved by multiple isomorphous replacement. The enzyme comprises two sandwiched beta sheets flanked by alpha-helical segments. The molecule shows internal symmetry, with the metal ions bound at the interface between the two halves. Despite less than 15% sequence identity, the protein fold resembles that of the catalytic domain of plant purple acid phosphatase and some serine/threonine protein phosphatases. The active-site regions of the mammalian and plant purple acid phosphatases differ significantly, however. The internal symmetry suggests that the binuclear centre evolved as a result of the combination of mononuclear ancestors. The structure of the mammalian enzyme provides a basis for antiosteoporotic drug design.

The Catalytic Mechanisms of Binuclear Metallohydrolases

Publisher: American Chemical Society (ACS)

Date: 06-2006

DOI: 10.1021/CR050318F

Structure-activity relationships for a new family of sulfonylurea herbicides

Publisher: Springer Science and Business Media LLC

Date: 11-2005

DOI: 10.1007/S10822-005-9028-9

Abstract: Acetohydroxyacid synthase (AHAS EC 2.2.1.6) catalyzes the first common step in branched-chain amino acid biosynthesis. The enzyme is inhibited by several chemical classes of compounds and this inhibition is the basis of action of the sulfonylurea and imidazolinone herbicides. The commercial sulfonylureas contain a pyrimidine or a triazine ring that is substituted at both meta positions, thus obeying the initial rules proposed by Levitt. Here we assess the activity of 69 monosubstituted sulfonylurea analogs and related compounds as inhibitors of pure recombinant Arabidopsis thaliana AHAS and show that disubstitution is not absolutely essential as exemplified by our novel herbicide, monosulfuron (2-nitro-N-(4'-methyl-pyrimidin-2'-yl) phenyl-sulfonylurea), which has a pyrimidine ring with a single meta substituent. A subset of these compounds was tested for herbicidal activity and it was shown that their effect in vivo correlates well with their potency in vitro as AHAS inhibitors. Three-dimensional quantitative structure-activity relationships were developed using comparative molecular field analysis and comparative molecular similarity indices analysis. For the latter, the best result was obtained when steric, electrostatic, hydrophobic and H-bond acceptor factors were taken into consideration. The resulting fields were mapped on to the published crystal structure of the yeast enzyme and it was shown that the steric and hydrophobic fields are in good agreement with sulfonylurea-AHAS interaction geometry.

Pyrrolidine nucleoside bisphosphonates as antituberculosis agents targeting hypoxanthine-guanine phosphoribosyltransferase

Publisher: Elsevier BV

Date: 11-2018

DOI: 10.1016/J.EJMECH.2018.09.039

Abstract: Therapeutic treatment of tuberculosis (TB) is becoming increasingly problematic due to the emergence of drug resistant Mycobacterium tuberculosis (Mt). Thus, new targets for anti-TB drug discovery need to be identified to combat and eradicate this disease. One such target is hypoxanthine-guanine phosphoribosyltransferase (HGPRT) which synthesises the 6-oxopurine nucleoside monophosphates essential for DNA/RNA production. [3R,4R]-4-Hypoxanthin-9-yl-3-((S)-2-hydroxy-2-phosphonoethyl)oxy-1-N-(phosphonopropionyl)pyrrolidine and [3R,4R]-4-guanin-9-yl-3-((S)-2-hydroxy-2-phosphonoethyl)oxy-1-N-(phosphonopropionyl)pyrrolidine (compound 6) are the most potent inhibitors of MtHGPRT yet discovered having K

International year of crystallography

Publisher: CSIRO Publishing

Date: 2014

DOI: 10.1071/CH14557

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.

Sources of pre-service teacher self-efficacy: a longitudinal qualitative inquiry

Publisher: Informa UK Limited

Date: 22-10-2022

DOI: 10.1080/02188791.2022.2136140

Structures of cell wall arabinosyltransferases with the anti-tuberculosis drug ethambutol

Publisher: American Association for the Advancement of Science (AAAS)

Date: 12-06-2020

DOI: 10.1126/SCIENCE.ABA9102

Abstract: Mycobacteria, including the species that causes tuberculosis (TB), synthesize a complex cell wall that helps to support and protect the bacterial cells. The major components of the cell wall include complex heteropolysaccharides that are synthesized in the periplasmic space. Zhang et al. determined the cryo–electron microscopy structures of two transmembrane glycosyltransferase enzyme complexes that use a lipid-anchored sugar donor to append arabinose units to the cell wall polysaccharides. They also captured the anti-TB drug ethambutol bound within these complexes and observed that it binds in a site overlapping both donor and acceptor sugars. Mapping of resistance mutants provides a structural understanding of how resistance emerges while preserving function of the enzyme and may help to guide the development of next-generation anti-TB drugs that target these enzymes. Science , this issue p. 1211

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

Aza-acyclic Nucleoside Phosphonates Containing a Second Phosphonate Group As Inhibitors of the Human, Plasmodium falciparum and vivax 6-Oxopurine Phosphoribosyltransferases and Their Pro

Publisher: American Chemical Society (ACS)

Date: 24-12-2014

DOI: 10.1021/JM501416T

Abstract: Hypoxanthine-guanine-[xanthine] phosphoribosyltransferase (HG[X]PRT) is considered an important target for antimalarial chemotherapy as it is the only pathway for the synthesis of the purine nucleoside monophosphates required for DNA/RNA production. Thus, inhibition of this enzyme should result in cessation of replication. The aza-acyclic nucleoside phosphonates (aza-ANPs) are good inhibitors of Plasmodium falciparum HGXPRT (PfHGXPRT), with Ki values as low as 0.08 and 0.01 μM for Plasmodium vivax HGPRT (PvHGPRT). Prodrugs of these aza-ANPs exhibit antimalarial activity against Pf lines with IC50 values (0.8-6.0 μM) and have low cytotoxicity against human cells. Crystal structures of six of these compounds in complex with human HGPRT have been determined. These suggest that the different affinities of these aza-ANPs could be due to the flexibility of the loops surrounding the active site as well as the flexibility of the inhibitors, allowing them to adapt to fit into three binding pockets of the enzyme(s).

Structure of CcmG/DsbE at 1.14 Å Resolution

Publisher: Elsevier BV

Date: 07-2002

DOI: 10.1016/S0969-2126(02)00794-3

Abstract: CcmG is unlike other periplasmic thioredoxin (TRX)-like proteins in that it has a specific reducing activity in an oxidizing environment and a high fidelity of interaction. These two unusual properties are required for its role in c-type cytochrome maturation. The crystal structure of CcmG reveals a modified TRX fold with an unusually acidic active site and a groove formed from two inserts in the fold. Deletion of one of the groove-forming inserts disrupts c-type cytochrome formation. Two unique structural features of CcmG-an acidic active site and an adjacent groove-appear to be necessary to convert an indiscriminately binding scaffold, the TRX fold, into a highly specific redox protein.

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.

Inhibition studies of ketol-acid reductoisomerases from pathogenic microorganisms

Publisher: Elsevier BV

Date: 10-2020

DOI: 10.1016/J.ABB.2020.108516

Commercial herbicides can trigger the oxidative inactivation of acetohydroxyacid synthase

Publisher: Wiley

Date: 29-02-2016

DOI: 10.1002/ANGE.201511985

Elucidating the Specificity of Binding of Sulfonylurea Herbicides to Acetohydroxyacid Synthase

Publisher: American Chemical Society (ACS)

Date: 28-01-2005

DOI: 10.1021/BI047980A

Abstract: Acetohydroxyacid synthase (AHAS, EC 2.2.1.6) is the target for the sulfonylurea herbicides, which act as potent inhibitors of the enzyme. Chlorsulfuron (marketed as Glean) and sulfometuron methyl (marketed as Oust) are two commercially important members of this family of herbicides. Here we report crystal structures of yeast AHAS in complex with chlorsulfuron (at a resolution of 2.19 A), sulfometuron methyl (2.34 A), and two other sulfonylureas, metsulfuron methyl (2.29 A) and tribenuron methyl (2.58 A). The structures observed suggest why these inhibitors have different potencies and provide clues about the differential effects of mutations in the active site tunnel on various inhibitors. In all of the structures, the thiamin diphosphate cofactor is fragmented, possibly as the result of inhibitor binding. In addition to thiamin diphosphate, AHAS requires FAD for activity. Recently, it has been reported that reduction of FAD can occur as a minor side reaction due to reaction with the carbanion/enamine of the hydroxyethyl-ThDP intermediate that is formed midway through the catalytic cycle. Here we report that the isoalloxazine ring has a bent conformation that would account for its ability to accept electrons from the hydroxyethyl intermediate. Most sequence and mutation data suggest that yeast AHAS is a high-quality model for the plant enzyme.

Synthesis of 9-phosphonoalkyl and 9-phosphonoalkoxyalkyl purines: Evaluation of their ability to act as inhibitors of Plasmodium falciparum, Plasmodium vivax and human hypoxanthine-guanine-(xanthine) phosphoribosyltransferases

Publisher: Elsevier BV

Date: 2012

DOI: 10.1016/J.BMC.2011.11.034

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.

Crystallization and preliminary X-ray analysis of a Kunitz-type inhibitor, textilinin-1 from Pseudonaja textilis textilis

Publisher: International Union of Crystallography (IUCr)

Date: 10-06-2006

DOI: 10.1107/S1744309106019099

Crystal structures of two novel sulfonylurea herbicides in complex with Arabidopsis thaliana acetohydroxyacid synthase

Publisher: Wiley

Date: 16-02-2009

DOI: 10.1111/J.1742-4658.2009.06863.X

Abstract: Acetohydroxyacid synthase (AHAS EC 2.2.1.6) is the first enzyme in the biosynthetic pathway of the branched-chain amino acids. It catalyzes the conversion of two molecules of pyruvate into 2-acetolactate or one molecule of pyruvate and one molecule of 2-ketobutyrate into 2-aceto-2-hydroxybutyrate. AHAS requires the cofactors thiamine diphosphate (ThDP), Mg(2+) and FAD for activity. The herbicides that target this enzyme are effective in protecting a broad range of crops from weed species. However, resistance in the field is now a serious problem worldwide. To address this, two new sulfonylureas, monosulfuron and monosulfuron ester, have been developed as commercial herbicides in China. These molecules differ from the traditional sulfonylureas in that the heterocyclic ring attached to the nitrogen atom of the sulfonylurea bridge is monosubstituted rather than disubstituted. The structures of these compounds in complex with the catalytic subunit of Arabidopsis thaliana AHAS have been determined to 3.0 and 2.8 A, respectively. In both complexes, these molecules are bound in the tunnel leading to the active site, such that the sole substituent of the heterocyclic ring is buried deepest and oriented towards the ThDP. Unlike the structures of Arabidopsis thaliana AHAS in complex with the classic disubstituted sulfonylureas, where ThDP is broken, this cofactor is intact and present most likely as the hydroxylethyl intermediate.

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.

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

Characterization and structural analysis of a potent anticoagulant phospholipase A2 from Pseudechis australis snake venom

Publisher: Elsevier BV

Date: 03-2016

DOI: 10.1016/J.TOXICON.2015.12.017

Abstract: Pseudechis australis is one of the most venomous and lethal snakes in Australia. Numerous phospholipase A2 (PLA2) isoforms constitute a major portion of its venom, some of which have previously been shown to exhibit not only enzymatic, but also haemolytic, neurotoxic and anticoagulant activities. Here, we have purified a potent anticoagulant PLA2 (identified as PA11) from P. australis venom to investigate its phospholipase, anticoagulant, haemolytic and cytotoxic activities and shown that addition of 11 nM PA11 resulted in a doubling of the clotting time of recalcified whole blood. We have also demonstrated that PA11 has high PLA2 enzymatic activity (10.9 × 10(4) Units/mg), but low haemolytic activity (0.6% of red blood cells hydrolysed in the presence of 1 nM PA11). PA11 at a concentration lower than 600 nM is not cytotoxic towards human cultured cells. Chemical modification experiments using p-bromophenacyl bromide have provided evidence that the catalytic histidine of PA11 is critical for the anticoagulant activity of this PLA2. PA11 that was subjected to trypsin digestion without previous reduction and alkylation of the disulfide bonds maintained enzymatic and anticoagulant activity, suggesting that proteolysis alone cannot abolish these properties. Consistent with these results, administration of PA11 by gavage in a rabbit stasis thrombosis model increased the clotting time of recalcified citrated whole blood by a factor of four. These data suggest that PA11 has potential to be developed as an anticoagulant in a clinical setting.

Synthesis of branched 9-[2-(2-phosphonoethoxy)ethyl]purines as a new class of acyclic nucleoside phosphonates which inhibit Plasmodium falciparum hypoxanthine–guanine–xanthine phosphoribosyltransferas

Publisher: Elsevier BV

Date: 09-2009

DOI: 10.1016/J.BMC.2009.07.044

Abstract: The malarial parasite Plasmodium falciparum (Pf) lacks the de novo pathway and relies on the salvage enzyme, hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT), for the synthesis of the 6-oxopurine nucleoside monophosphates. Specific acyclic nucleoside phosphonates (ANPs) inhibit PfHGXPRT and possess anti-plasmodial activity. Two series of novel branched ANPs derived from 9-[2-(2-phosphonoethoxy)ethyl]purines were synthesized to investigate their inhibition of PfHGXPRT and human HGPRT. The best inhibitor of PfHGXPRT has a K(i) of 1 microM. The data showed that both the position and nature of the hydrophobic substituent change the potency and selectivity of the ANPs.

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

Publisher: Wiley

Date: 07-2020

DOI: 10.1002/CHEM.202000899

Crystallization and preliminary diffraction studies of native and selenomethionine CcmG (CycY, DsbE)

Publisher: International Union of Crystallography (IUCr)

Date: 23-08-2001

DOI: 10.1107/S0907444901009982

Abstract: Disulfide-bond (Dsb) proteins are a family of redox proteins containing a Cys-X-X-Cys motif. They are essential for disulfide-bond exchange in the bacterial periplasm and are necessary for the correct folding and function of many secreted proteins. CcmG (DsbE) is a reducing Dsb protein required for cytochrome c maturation. Crystals of Bradyrhizobium japonicum CcmG have been obtained that diffract X-rays to 1.14 A resolution. The crystals are orthorhombic, space group P2(1)2(1)2(1), with unit-cell parameters a = 35.1, b = 48.2, c = 90.2 A. Selenomethionine CcmG was expressed without using a methionine auxotroph or methionine-pathway inhibition and was purified without reducing agents.

University Of Utah

Location: United States of America

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Harrington Cancer Center

Location: United States of America

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Saint Vincent's Institute Of Medical Research

Location: Australia

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Monash University

Location: Australia

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University Of Queensland

Location: Australia

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Linkage Projects - Grant ID: LP0454264

Start Date: 06-2004

End Date: 06-2007

Amount: $888,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP150104358

Start Date: 2015

End Date: 06-2019

Amount: $429,100.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0208682

Start Date: 2002

End Date: 12-2004

Amount: $155,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0450275

Start Date: 2004

End Date: 12-2007

Amount: $210,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE230100145

Start Date: 05-2023

End Date: 04-2024

Amount: $1,129,423.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP210101802

Start Date: 08-2021

End Date: 08-2024

Amount: $690,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP0882340

Start Date: 01-2008

End Date: 12-2012

Amount: $788,533.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0558652

Start Date: 2005

End Date: 06-2008

Amount: $420,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0986292

Start Date: 2009

End Date: 07-2012

Amount: $350,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE200100140

Start Date: 03-2020

End Date: 06-2021

Amount: $945,000.00

Funder: Australian Research Council