Structure-Based Design Approaches to Cell Wall Biosynthesis Inhibitors

Katz, Alfred; Caufield, Craig E.

doi:10.2174/1381612033455305

Cited by 63 publications

(33 citation statements)

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“…Isomorphous cocrystals of the LL-AziDAP-and DLAziDAP-inhibited forms of DAP epimerase belong to space group C222 1 . Both structures were solved by molecular replacement and refined to respectable R factors.…”

Section: Resultsmentioning

confidence: 99%

Structural insights into stereochemical inversion by diaminopimelate epimerase: An antibacterial drug target

Pillai

Cherney

Diaper

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

113

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D-amino acids are much less common than their L-isomers but are widely distributed in most organisms. Many D-amino acids, including those necessary for bacterial cell wall formation, are synthesized from the corresponding L-isomers by ␣-amino acid racemases. The important class of pyridoxal phosphate-independent racemases function by an unusual mechanism whose details have been poorly understood. It has been proposed that the stereoinversion involves two active-site cysteine residues acting in concert as a base (thiolate) and an acid (thiol). Although crystallographic structures of several such enzymes are available, with the exception of the recent structures of glutamate racemase from Bacillus subtilis and of proline racemase from Trypanosoma cruzi, the structures either are of inactive forms (e.g., disulfide) or do not allow unambiguous modeling of the substrates in the active sites. Here, we present the crystal structures of diaminopimelate (DAP) epimerase from Haemophilus influenzae with two different isomers of the irreversible inhibitor and substrate mimic aziridino-DAP at 1.35-and 1.70-Å resolution. These structures permit a detailed description of this pyridoxal 5 -phosphate-independent amino acid racemase active site and delineate the electrostatic interactions that control the exquisite substrate selectivity of DAP epimerase. Moreover, the active site shows how deprotonation of the substrates' nonacidic hydrogen at the ␣-carbon (pKa Ϸ 29) by a seemingly weakly basic cysteine residue (pK a Ϸ 8 -10) is facilitated by interactions with two buried ␣-helices. Bacterial racemases, including glutamate racemase and DAP epimerase, are potential targets for the development of new agents effective against organisms resistant to conventional antibiotics.racemase ͉ stereochemical mechanism T he increase in microbial resistance to conventional antibiotics has rekindled intense interest in new methods of inhibiting bacterial cell wall biosynthesis (1), including blocking formation or utilization of D-amino acids such as D-alanine (2), D-glutamate (3), and meso-diaminopimelic acid (4) present in peptidoglycan (5). The inclusion of D-amino acids in the peptidoglycan layer of the cell wall is thought to provide bacteria with protection from the action of host proteases (2). Glutamate racemase (6, 7) and diaminopimelate (DAP) epimerase (EC 5.1.1.7) (8), as well as aspartate racemase (9) and proline racemase (10, 11), are examples of pyridoxal 5Ј-phosphate (PLP)-independent amino acid racemases that invert the configuration at the ␣-carbon of an amino acid without the use of cofactors, metals, or reducible keto or imino functionalities. The proposed ''two-base'' mechanism (11) for these enzymes involves one active-site cysteine thiolate acting as a base to deprotonate the ␣-carbon, while a second cysteine thiol functions as an acid to reprotonate the resulting planar carbanionic intermediate from the opposite face (Scheme 1). This process is not trivial, because the pK a of the ␣-proton is Ϸ21 for the fully protonated fo...

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Section: Resultsmentioning

confidence: 99%

Structural insights into stereochemical inversion by diaminopimelate epimerase: An antibacterial drug target

Pillai

Cherney

Diaper

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

113

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“…The subsequent steps in the pathway are entirely dependent on the MurB product UDP-MurNAc for completion and delivery of peptidoglycan monomer units to actively growing regions of the bacterial cell wall. Due to its importance in peptidoglycan biosynthesis, the MurB enzyme has been the subject of a number of target-based drug design efforts (8,18,21,36,39,69).…”

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confidence: 99%

Evaluation of the metS and murB Loci for Antibiotic Discovery Using Targeted Antisense RNA Expression Analysis in Bacillus anthracis

Kedar

Brown-Driver

Reyes

et al. 2007

Antimicrob Agents Chemother

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The biowarfare-relevant bacterial pathogen Bacillus anthracis contains two paralogs each of the metS and murB genes, which encode the important antibiotic target functions methionyl-tRNA synthetase and UDP-Nacetylenolpyruvoylglucosamine reductase, respectively. Empirical screens were conducted to detect and characterize gene fragments of each of these four genes that could cause growth reduction of B. anthracis when inducibly expressed from a plasmid-borne promoter. Numerous such gene fragments that were overwhelmingly in the antisense orientation were identified for the metS1 and murB2 alleles, while no such orientation bias was seen for the metS2 and murB1 alleles. Gene replacement mutagenesis was used to confirm the essentiality of the metS1 and murB2 alleles, and the nonessentiality of the metS2 and murB1 alleles, for vegetative growth. Induced transcription of RNA from metS1 and murB2 antisense-oriented gene fragments resulted in specific reduction of mRNA of their cognate genes. Attenuation of MetS1 enzyme expression hypersensitized B. anthracis cells to a MetS-specific antimicrobial compound but not to other antibiotics that affect cell wall assembly, fatty acid biosynthesis, protein translation, or DNA replication. Antisense-dependent reduction of MurB2 enzyme expression caused hypersensitivity to beta-lactam antibiotics, a synergistic response that has also been noted for the MurA-specific antibiotic fosfomycin. These experiments form the basis of mode-ofaction detection assays that can be used in the discovery of novel MetS-or MurB-specific antibiotic drugs that are effective against B. anthracis or other gram-positive bacterial pathogens.

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“…The absence of a homologue in eukaryotic cells makes MurB an attractive target for small molecule inhibitors with the potential to have broad antibacterial activity. Biochemical characterization and X-ray structural analysis of MurB from Escherichia coli (3-5, 18, 45), S. aureus (6), and Streptococcus pneumoniae (49) have been published and have been used in structure-based approaches to design inhibitors (30). Based on a cocrystal structure study of E. coli MurB with enolpyruvyl-UDP-N-acetylglucosamine (EP-UNAG) (3,4), it was found that the carboxylate of the substrate interacts with residues Arg159 and Glu325 and may be responsible for transition state stabilization, whereas the diphosphate moiety of the substrate interacts with residues Tyr190, Lys217, Asn233, and Glu288.…”

mentioning

confidence: 99%

3,5-Dioxopyrazolidines, Novel Inhibitors of UDP- N - Acetylenolpyruvylglucosamine Reductase (MurB) with Activity against Gram-Positive Bacteria

Yang¹,

Severin²,

Chopra³

et al. 2006

Antimicrob Agents Chemother

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Structure-Based Design Approaches to Cell Wall Biosynthesis Inhibitors

Cited by 63 publications

References 0 publications

Structural insights into stereochemical inversion by diaminopimelate epimerase: An antibacterial drug target

Structural insights into stereochemical inversion by diaminopimelate epimerase: An antibacterial drug target

Evaluation of the metS and murB Loci for Antibiotic Discovery Using Targeted Antisense RNA Expression Analysis in Bacillus anthracis

3,5-Dioxopyrazolidines, Novel Inhibitors of UDP- N - Acetylenolpyruvylglucosamine Reductase (MurB) with Activity against Gram-Positive Bacteria

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