RESUMO
Inhibitors of the ATPase function of bacterial DNA gyrase, located in the GyrB subunit and its related ParE subunit in topoisomerase IV, have demonstrated antibacterial activity. In this study we describe an NMR fragment-based screening effort targeting Staphylococcus aureus GyrB that identified several attractive and novel starting points with good ligand efficiency. Fragment hits were further characterized using NMR binding studies against full-length S. aureus GyrB and Escherichia coli ParE. X-ray co-crystal structures of select fragment hits confirmed binding and suggested a path for medicinal chemistry optimization. The identification, characterization, and elaboration of one of these fragment series to a 0.265 µM inhibitor is described herein.
Assuntos
Antibacterianos/química , Proteínas de Bactérias/antagonistas & inibidores , DNA Girase/química , Inibidores da Topoisomerase II/química , Adenosina Trifosfatases/metabolismo , Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , DNA Girase/metabolismo , DNA Topoisomerase IV/antagonistas & inibidores , DNA Topoisomerase IV/metabolismo , Desenho de Fármacos , Escherichia coli/metabolismo , Ligantes , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Staphylococcus aureus/enzimologia , Inibidores da Topoisomerase II/metabolismoRESUMO
The transfer of the phosphopantetheine chain from coenzyme A (CoA) to the acyl carrier protein (ACP), a key protein in both fatty acid and polyketide synthesis, is catalyzed by ACP synthase (AcpS). Streptomyces coelicolor AcpS is a doubly promiscuous enzyme capable of activation of ACPs from both fatty acid and polyketide synthesis and catalyzes the transfer of modified CoA substrates. Five crystal structures have been determined, including those of ligand-free AcpS, complexes with CoA and acetyl-CoA, and two of the active site mutants, His110Ala and Asp111Ala. All five structures are trimeric and provide further insight into the mechanism of catalysis, revealing the first detailed structure of a group I active site with the essential magnesium in place. Modeling of ACP binding supported by mutational analysis suggests an explanation for the promiscuity in terms of both ACP partner and modified CoA substrates.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Modelos Moleculares , Streptomyces coelicolor/enzimologia , Transferases (Outros Grupos de Fosfato Substituídos)/química , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Domínio Catalítico/genética , Cristalografia por Raios X , Proteínas de Escherichia coli/química , Ligantes , Magnésio/química , Dados de Sequência Molecular , Ligação Proteica/genética , Dobramento de Proteína , Streptomyces coelicolor/genética , Especificidade por Substrato/genética , Transferases/química , Transferases (Outros Grupos de Fosfato Substituídos)/genéticaRESUMO
Sperm glyceraldehyde-3-phosphate dehydrogenase has been shown to be a successful target for a non-hormonal contraceptive approach, but the agents tested to date have had unacceptable side effects. Obtaining the structure of the sperm-specific isoform to allow rational inhibitor design has therefore been a goal for a number of years but has proved intractable because of the insoluble nature of both native and recombinant protein. We have obtained soluble recombinant sperm glyceraldehyde-3-phosphate dehydrogenase as a heterotetramer with the Escherichia coli glyceraldehyde-3-phosphate dehydrogenase in a ratio of 1:3 and have solved the structure of the heterotetramer which we believe represents a novel strategy for structure determination of an insoluble protein. A structure was also obtained where glyceraldehyde 3-phosphate binds in the P(s) pocket in the active site of the sperm enzyme subunit in the presence of NAD. Modeling and comparison of the structures of human somatic and sperm-specific glyceraldehyde-3-phosphate dehydrogenase revealed few differences at the active site and hence rebut the long presumed structural specificity of 3-chlorolactaldehyde for the sperm isoform. The contraceptive activity of alpha-chlorohydrin and its apparent specificity for the sperm isoform in vivo are likely to be due to differences in metabolism to 3-chlorolactaldehyde in spermatozoa and somatic cells. However, further detailed analysis of the sperm glyceraldehyde-3-phosphate dehydrogenase structure revealed sites in the enzyme that do show significant difference compared with published somatic glyceraldehyde-3-phosphate dehydrogenase structures that could be exploited by structure-based drug design to identify leads for novel male contraceptives.
Assuntos
Anticoncepção , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/química , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Espermatozoides/enzimologia , Sequência de Aminoácidos , Animais , Cromatografia por Troca Iônica , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Proteínas de Escherichia coli/genética , Feminino , Gliceraldeído-3-Fosfato Desidrogenases/genética , Humanos , Masculino , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Homologia de Sequência de Aminoácidos , Homologia Estrutural de ProteínaRESUMO
Neisseria meningitidis is an obligate commensal bacterium of humans, and also an important human pathogen. To facilitate future drug studies, we report here the biochemical and structural characterisation of a key enzyme in (S)-lysine biosynthesis, dihydrodipicolinate synthase (DHDPS), from N. meningitidis (NmeDHDPS). X-ray crystallography revealed only minor structural differences between NmeDHDPS and the enzyme from E. coli at the active and allosteric effector sites. The catalytic capabilities of NmeDHDPS are similar to those of the enzyme from E. coli, but intriguingly NmeDHDPS is subject to substrate inhibition by high concentrations of the second substrate, (S)-aspartate semialdehyde, and is also significantly more sensitive to feedback inhibition by (S)-lysine. This heightened sensitivity to inhibition at both active and allosteric sites suggests that it may be possible to target DHDPS from N. meningitidis for antibiotic development.
Assuntos
Hidroliases/metabolismo , Neisseria meningitidis/enzimologia , Dicroísmo Circular , Clonagem Molecular , Cristalografia por Raios X , Estabilidade Enzimática , Escherichia coli/enzimologia , Hidroliases/química , Hidroliases/genética , Hidroliases/isolamento & purificação , Ligação de Hidrogênio , Cinética , Multimerização ProteicaRESUMO
Maleyl pyruvate isomerase (MPI) is a bacterial glutathione S-transferase (GST) from the pathway for degradation of naphthalene via gentisate that enables the bacterium Ralstonia to use polyaromatic hydrocarbons as a sole carbon source. Genome sequencing projects have revealed the presence of large numbers of GSTs in bacterial genomes, often located within gene clusters encoding the degradation of different aromatic compounds. This structure is therefore an example of this under-represented class of enzymes. Unlike many glutathione transferases, the reaction catalysed by MPI is an isomerisation of an aromatic ring breakdown product, and glutathione is a true cofactor rather than a substrate in the reaction. We have solved the structure of the enzyme in complex with dicarboxyethyl glutathione, an analogue of a proposed reaction intermediate, at a resolution of 1.3 A. The structure provides direct evidence that the glutathione thiolate attacks the substrate in the C2 position, with the terminal carboxylate buried at the base of the active site cleft. Our structures suggest that the C1-C2 bond remains fixed so when rotation occurs around the C2-C3 bond the atoms from C4 onwards actually move. We identified a conserved arginine that is likely to stabilize the enolate form of the substrate during the isomerisation. Arginines at either side of the active site cleft can interact with the end of the substrate/product and preferentially stabilise the product. MPI has significant sequence similarity to maleylacetoacetate isomerase (MAAI), which performs an analogous reaction in the catabolism of phenylalanine and tyrosine. The proposed mechanism therefore has relevance to the MAAIs. Significantly, whilst the overall sequence identity is 40% only one of the five residues from the Zeta motif in the active site is conserved. We re-examined the roles of the residues in the active site of both enzymes and the Zeta motif itself.
Assuntos
Glutationa Transferase/química , Ralstonia/enzimologia , cis-trans-Isomerases/química , Sequência de Aminoácidos , Arabidopsis/enzimologia , Sítios de Ligação , Cristalografia por Raios X , Ácidos Dicarboxílicos/metabolismo , Dimerização , Glutationa/metabolismo , Glutationa Transferase/metabolismo , Humanos , Isomerismo , Modelos Moleculares , Dados de Sequência Molecular , Naftalenos/metabolismo , Ácidos Pimélicos/metabolismo , Dobramento de Proteína , Estrutura Secundária de Proteína , Alinhamento de Sequência , cis-trans-Isomerases/metabolismoRESUMO
Metallo-beta-lactamases (mbetals) confer broad-spectrum resistance to beta-lactam antibiotics upon host bacteria and escape the action of existing beta-lactamase inhibitors. SPM-1 is a recently discovered mbetal that is distinguished from related enzymes by possession of a substantial central insertion and by sequence variation at positions that maintain active site structure. Biochemical data show SPM-1 to contain two Zn2+ sites of differing affinities, a phenomenon that is well documented amongst mbetals but for which a structural explanation has proved elusive. Here, we report the crystal structure of SPM-1 to 1.9 A resolution. The structure reveals SPM-1 to lack a mobile loop implicated in substrate binding by related mbetals and to accommodate the central insertion in an extended helical interdomain region. Deleting this had marginal effect upon binding and hydrolysis of a range of beta-lactams. These data suggest that the interactions of SPM-1 with substrates differ from those employed by other mbetals. SPM-1 as crystallised contains a single Zn2+. Both the active site hydrogen-bonding network and main-chain geometry at Asp120, a key component of the binding site for the second zinc ion, differ significantly from previous mbetal structures. We propose that variable interactions made by the Asp120 carbonyl group modulate affinity for a second Zn2+ equivalent in mbetals of the B1 subfamily. We further predict that SPM-1 possesses the capacity to evolve variants of enhanced catalytic activity by point mutations altering geometry and hydrogen bonding in the vicinity of the second Zn2+ site.
Assuntos
Pseudomonas aeruginosa/enzimologia , Zinco/metabolismo , beta-Lactamases/química , beta-Lactamases/metabolismo , Sítios de Ligação/genética , Domínio Catalítico/genética , Cristalização , Cristalografia por Raios X , Dimerização , Estrutura Quaternária de Proteína/genética , Estrutura Terciária de Proteína/genética , Pseudomonas aeruginosa/genética , Zinco/química , beta-Lactamases/genéticaRESUMO
Unsaturated and fluorinated analogues of aspartyl-beta-phosphate were synthesised as potential inhibitors of the bacterial enzyme aspartate semialdehyde dehydrogenase (ASA-DH). Acetylenic and Z-olefinic analogues showed competitive inhibition, but an E-olefinic analogue was inactive. A monofluoromethylene phosphonate competed poorly, but showed time-dependent inhibition of ASA-DH in the absence of phosphate. Simulated docking procedures were used to rationalise the results. These studies showed that substrate and inhibitor binding are mediated by interaction with two active-site arginine residues, and for likely covalent attachment to the active-site thiol group, electrophilic carbon atoms should be located 4.5 A, or less, from the thiol.
Assuntos
Aspartato-Semialdeído Desidrogenase/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Arginina , Ácido Aspártico/análogos & derivados , Ácido Aspártico/síntese química , Ácido Aspártico/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Sítios de Ligação , Ligação Competitiva , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
We have determined the 2.5 angstroms crystal structure of an active, tetrameric Streptomyces coelicolor type II polyketide ketoreductase (actIII) with its bound cofactor, NADP+. This structure shows a Rossman dinucleotide binding fold characteristic of SDR enzymes. Of two subunits in the crystallographic asymmetric unit, one is closed around the active site. Formate is observed in the open subunit, indicating possible carbonyl binding sites of the polyketide intermediate. Unlike previous models we observe crystal contacts that may mimic the KR-ACP interactions that may drive active site opening. Based on these observations, we have constructed a model for ACP and polyketide binding. We propose that binding of ACP triggers a conformational change from the closed to the open, active form of the enzyme. The polyketide chain enters the active site and reduction occurs. The model also suggests a general mechanism for ACP recognition which is applicable to a range of protein families.
Assuntos
Proteína de Transporte de Acila/metabolismo , Oxirredutases do Álcool/química , Oxirredutases do Álcool/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Modelos Moleculares , Sequência de Aminoácidos , Sítios de Ligação , Cristalização , Cristalografia , Formiatos/química , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Subunidades Proteicas , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
Polyketide metabolites produced by bacteria and other organisms include antibiotics, anticancer and antifungal compounds. In type II polyketide synthesis, three enzymes are sufficient to form a polyketide product of the requisite chain length, although the fidelity of the first cyclization is variable. Addition of ketoreductase (KR) to this system results in the formation of a product with correct cyclization and reduction. This paper reports the cloning of the Streptomyces coelicolor actIII ORF5 gene that codes for the ketoreductase. The 261-amino-acid protein has been overexpressed with a 20-residue His tag, purified by affinity chromatography and crystallized in space group P3(2)21, with unit-cell parameters a = b = 103.9, c = 123.1 angstroms. The crystals diffract to 2.5 angstroms resolution. A complete data set has been collected and structure solution and refinement is under way.