Synthesis and evaluation of inhibitors of bacterial d-alanine: d-alanine ligases

Ellsworth, Bruce A.; Tom, Norma J.; Bartlett, Paul A.

doi:10.1016/s1074-5521(96)90082-4

Cited by 69 publications

(68 citation statements)

References 23 publications

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“…The tight binding of D-cycloserine to DDl suggests that the antibiotic binds to the high-affinity D-alanine site (10). Compounds designed to mimic the Dalanine:D-alanine dipeptide or the reaction transition-state intermediates led to the developments of phosphinate and phosphonate dipeptide analogs (11)(12). These phosphinates or phosphonates, after being phosphorylated by DDl, bind to the protein tightly and inhibit the reaction (11)(12).…”

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confidence: 99%

“…Compounds designed to mimic the Dalanine:D-alanine dipeptide or the reaction transition-state intermediates led to the developments of phosphinate and phosphonate dipeptide analogs (11)(12). These phosphinates or phosphonates, after being phosphorylated by DDl, bind to the protein tightly and inhibit the reaction (11)(12). The best in vitro IC 50 of these compounds are Ϸ4 M on Streptococcus faecalis DDl, and their antibacterial activities are not significant (11).…”

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Allosteric inhibition of Staphylococcus aureus d -alanine: d -alanine ligase revealed by crystallographic studies

Liu

Chang

Herberg

et al. 2006

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

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Allosteric inhibition of Staphylococcus aureus d -alanine: d -alanine ligase revealed by crystallographic studies

Liu

Chang

Herberg

et al. 2006

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

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“…The enzymes have similar functions in the biosynthesis of peptidoglycan termini in bacteria, but the capacity of VanA to form D-Ala-D-Lac has a direct correlation with enterococcal resistance to vancomycin. In this paper, we report the crystal structure of VanA complexed with a substrate inhibitor (7) and define the residues involved in substrate binding that are crucial to the resistance mechanism.…”

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confidence: 99%

The molecular basis of vancomycin resistance in clinically relevant Enterococci: Crystal structure of d -alanyl- d -lactate ligase (VanA)

Roper

Huyton

Vagin

et al. 2000

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

107

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D-alanine-D-lactate ligase from Enterococcus faecium BM4147 is directly responsible for the biosynthesis of alternate cell-wall precursors in bacteria, which are resistant to the glycopeptide antibiotic vancomycin. The crystal structure has been determined with data extending to 2.5-Å resolution. This structure shows that the active site has unexpected interactions and is distinct from previous models for D-alanyl-D-lactate ligase mechanistic studies. It appears that the preference of the enzyme for lactate as a ligand over D-alanine could be mediated by electrostatic effects and͞or a hydrogen-bonding network, which principally involve His-244. The structure of D-alanyl-D-lactate ligase provides a revised interpretation of the molecular events that lead to vancomycin resistance. The first step in defining the structural and biochemical basis of vancomycin resistance was the determination of the structure of the enzyme [D-Ala-D-Ala ligase (DdlB), from Escherichia coli] that synthesizes the D-Ala-D-Ala moiety incorporated into the normal vancomycin-susceptible cell walls (6). The sequence of VanA and DdlB exhibits Ϸ40% identity, and their overall folds are very similar. The enzymes have similar functions in the biosynthesis of peptidoglycan termini in bacteria, but the capacity of VanA to form D-Ala-D-Lac has a direct correlation with enterococcal resistance to vancomycin. In this paper, we report the crystal structure of VanA complexed with a substrate inhibitor (7) and define the residues involved in substrate binding that are crucial to the resistance mechanism. Materials and MethodsPurification and Crystallization. The purification and crystallization of the enzyme have been described in detail elsewhere (8). Briefly, the enzyme was crystallized by the hanging-drop-vapor diffusion method in the presence of a phosphinate inhibitor analogue of D-Ala-D-Ala between 40% and 45% ammonium sulfate, 0.1 M Mops, pH 6.0, with crystals reaching dimensions of 0.4 ϫ 0.2 ϫ 0.1 mm. A native dataset to 2.5 Å resolution was collected and the space group determined as C222 1 with cell dimensions of a ϭ 123.2 Å, b ϭ 225 Å, and c ϭ 72.4 Å, with a single dimer in the asymmetric unit. Data were collected with an oscillation range of 1°per image by using a Cu-K a radiation source and a MAR345 image plate device. Crystals were soaked in mother liquor containing a glycerol concentration of 35% as cryoprotectant before flash vitrification in a stream of N 2 gas at 120 K. The data were processed and reduced by using the HKL suite of programs (9).Structure Determination and Refinement. Numerous attempts to solve the structure of VanA by molecular replacement by using a variety of programs had proved unsuccessful until recent in-house developments in the molecular replacement program MOLREP (10) became available. A solution was obtained by using This paper was submitted directly (Track II) to the PNAS office.

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“…The monomethyl ester 9 was obtained by partial base-catalyzed hydrolysis of dimethyl ester 8 and was subsequently converted by treatment with thionyl chloride in dichloromethane to chlorophosphonate 10. This strategy revealed the key intermediate chlorophosphonate 10 could be coupled with d-lactic acid methyl ester or d-alanine methyl ester and converted using previously published procedures 8,10,14 to aord the phosphonate 3 and phosphonamidate 1. Similar synthetic Schemes are thoroughly precedented in the literature.…”

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“…The crystal structure of VanX demonstrated that the comparable N±H in substrate d-Ala-d-Ala is hydrogen bonded to the backbone carbonyl of Tyr109. 6 To extend the synthetic route in Scheme 1 to generate additional potential inhibitors of VanX, chlorophosphonate 10 was coupled with methyl mercaptoacetate and deprotected using standard procedures 8,10,14 to yield the phosphothioate 2. This compound was shown to be stable for over a week at 4 C and at pH 7.0.…”

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Phosphonamidate and phosphothioate dipeptides as potential inhibitors of VanX

Yang

Brandt

Chatwood

et al. 2000

Bioorganic & Medicinal Chemistry Letters

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Synthesis and evaluation of inhibitors of bacterial d-alanine: d-alanine ligases

Cited by 69 publications

References 23 publications

Allosteric inhibition of Staphylococcus aureus d -alanine: d -alanine ligase revealed by crystallographic studies

Allosteric inhibition of Staphylococcus aureus d -alanine: d -alanine ligase revealed by crystallographic studies

The molecular basis of vancomycin resistance in clinically relevant Enterococci: Crystal structure of d -alanyl- d -lactate ligase (VanA)

Phosphonamidate and phosphothioate dipeptides as potential inhibitors of VanX

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