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FIG. 1. (A) Vancomycin binds the D-Ala-D-Ala moiety of the growing peptidoglycan and sterically occludes the transglycosylation and transpeptidation steps of cell-wall assembly. The immature cell wall results in cells susceptible to lysis through osmotic shock. (B) The alternative cell-wall biosynthetic pathway of the VanH, -A, -X proteins, producing peptidoglycan intermediates with D-Ala-D-lactate termini in place of the usual D-Ala-D-Ala termini (12). Pyruvate is reduced to D-lactate by the NADP-dependent dehydrogenase VanH, which is then used as substrate for the ATP-dependent D-Ala-D-lactate depsipeptide ligase VanA. The product D-Ala-D-lactate depsipeptide is used by the enzyme MurF to produce the muramyl-peptidyl-D-lactate intermediate and brought forward in subsequent cell-wall biosynthesis. The zinc-dependent D, D-dipeptidase VanX, specifically hydrolyzes the D-Ala-D-Ala dipeptide pool produced by the native D-Ala-D-Ala Ddl ligase without hydrolyzing the D-Ala-D-lactate and in this way effectively shunts the flux of the cell-wall biosynthesis to the ester termini. Substitution of D-Ala by D-lactate does not impair crosslinking of the modified precursors to the growing peptidoglycan chain, resulting in a mechanically strong peptidoglycan layer and cell survival. (C) Structures of the vancomycin complexes with N-acyl-D-Ala-D-Ala and N-acyl-D-Ala-D-lactate (9). Vancomycin binds to the D-Ala-D-Ala termini through a five-hydrogen bond network. The key hydrogen bond between the D-Ala amide NH and the vancomycin backbone carbonyl is lost in the N-acyl-D-Ala-D-lactate complex, resulting in a 1,000-fold reduction in the affinity of the antibiotic.

Table 1. VanX homologs

VanX source

Role

Vancomycin-resistant

enterococci

Enterococcus faecium

Enterococcus faecalis

Reprogram cell walls for vancomycin resistance in opportunistic pathogens

Glycopeptide producers

Streptomyces

toyocaensis

Amycolatopsis orientalis

Coevolution of vanHAX operon with antibiotic biosynthesis genes for immunity

Stationary-phase survival

mechanism

Escherichia coli

Transport D-Ala-D-Ala from periplasm back to cytoplasm as cell-wall crosslinks are refashioned and use as RpoS-mediated energy source

 

medicinal chemistry challenge. The likely mechanism for D, D-dipeptide hydrolysis, is shown in Fig. 2C with Glu-181 acting as catalytic base and Arg-71 as a cationic coordinator both in the ground state and for stabilization of the developing negative charge in the tetrahedral adduct (18, 20). The structure predicted Asp-123, Asp-142, and Tyr-21 residues for the recognition of the D-Ala-D-Ala dipeptide substrate a-NH3+ and Ser-114 for the carboxylate group. Site-directed mutagenesis of active site residues has revealed roles consistent with predictions of recognition and catalysis (20).

VanX Homologs in the Bacteria That Produce Vancomycin and Related Glycopeptide Antibiotics. In many instances, bacteria that produce antibiotics have evolved strategies and mechanisms that provide immunity to the action of the antibiotic, and there is a general supposition that immunity mechanisms will have coevolved with antibiotic biosynthesis genes to protect the producing organisms (21, 22). Streptomyces toyocaensis synthesizes and secretes a vancomycin-type glycopeptide antibiotic (A47934), and the molecular basis of immunity for this organism and most likely for Amylocatopsis orientalis, which produces vancomycin, has been recently deconvolved (14, 15, 23, 24). PCR probes to EntVanX zinc-binding motif revealed an S.toyocaensis VanX homologue (StoVanX) with 63% similarity to EntVanX, and sequencing analysis then indicated a three-gene operon in S.toyocaensis and A.orientalis equivalent and similarly oriented to the vanHAX operon from (Fig. 3A). Expression and purification of the StoVanX confirms it is a high efficiency D,D-dipeptidase with unmodified N and C termini and that it. lacks D-Ala-D-lactate depsipeptide activity (15) (Table 2). These findings suggest a conserved mechanism for the observed intrinsic resistance of the antibiotic producers to the vancomycin class of glycopeptides and that before S.toyocaensis produces the glycopeptide A47934, it has D-Ala-D-Ala peptidoglycan termini and is sensitive to vancomycin-type antibiotics. Furthermore, S.toyocaensis possesses two D-, D-ligases: a D-Ala-D-lactate ligase encoded by the vanHAX operon and a D-Ala-D-Ala ligase encoded by a separate gene on the chromosome (24). The S.toyocaensis vanHAX equivalents may be switched on transcriptionally when the host turns on the cluster of genes to synthesize the glycopeptide A47934 to reprogram PG

   

N-acyl-D-Ala-D-lactate (9). Vancomycin binds to the D-Ala-D-Ala termini through a five-hydrogen bond network. The key hydrogen bond between the D-Ala amide NH and the vancomycin backbone carbonyl is lost in the N-acyl-D-Ala-D-lactate complex, resulting in a 1,000-fold reduction in the affinity of the antibiotic.



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