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FIG. 2. (A) Structure of EntVanX (18). (B) Active site topology of EntVanX complex with the phosphinate analog (18). The zinc atom is coordinated with His-116, Asp-123, and His-184. The phosphinate analog a-NH3+ hydrogen bonds with Asp-123, Asp-142, and Tyr-21, whereas Ser-114 hydrogen bonds with the carboxylate group. Arg-71 stabilizes the transition state intermediate, represented by the phosphinate analog. Glu-181 is the catalytic base. (C) Proposed mechanism of VanX (20). The water molecule is activated by Glu-181 and attacks the zinc-polarized carbonyl to form a tetrahedral adduct, which is then stabilized by both the zinc atom and the Arg-71. The Glu-181 transfers the proton to the nitrogen, which is hydrogen bonded to the carbonyl group of Tyr-109; peptide bond cleavage follows [C; reprinted from ref. 20 with kind permission from Elsevier Science (Amsterdam)].

termini to end in D-AlaD-lactate, providing in situ resistance to the produced antibiotic (Fig. 3B). This three-gene operon has also been detected in other glycopeptide-producing organisms (14), and one of these operons may have been the origin for the enteroccocal vanHAX genes that are on trans

Table 2. Catalytic efficiencies of VanX homologs on zinc-dependent D-AlaD-Ala dipeptidases (15)

VanX enzymes

Mol % zinc content

KMµM

kcat s–1

kcat/KM s–1mM–1

EntVanX

(Enterococcus faecalis)

95

80

26

325

StoVanX

(Streptomyces toyocaensis)

84

4

12

3,000

DdpX

(Escherichia coli)

100

14,000

170

12

Substrate specificity:

*D-, D-dipeptides with unmodified N or C termini,

*Does not hydrolyze esters, tripeptides, or dipeptides of L/L or mixed diastereomeric configuration (L/D or D/L).

posable elements in most of the VanA clinical phenotypes of vancomycin-resistant enterococci (VRE). Noticeably, the G+ C content of the vanHAX operon in VRE is 5–10% higher than the adjacent vanSR genes and chromosomal genes of enterococci. These findings also exemplify a mechanism for coevolution of glycopeptide antibiotic production and glycopeptide antibiotic resistance, the latter then appropriated by the opportunistic pathogenic enterococci.

The Dilemma for E.coli Strains That Contain and Express the VanX Homolog (ddpX). Analysis of the E.coli genome database turned up a possible VanX homologue [originally referred to as EcoVanX (15) and renamed here DdpX] with 27% similarity to EntVanX. Expression and purification validated the expected activity, although the KM of 14 mM for D-AlaD-Ala was 250- to 3,000-fold elevated compared with the EntVanX and StoVanX enzymes (15), consistent with a purely degradative function for the DdpX (Table 2). All of the active site residues and auxiliary residues that maintain the active-site topology in EntVanX are conserved in DdpX, and kinetic analysis also revealed the same substrate specificity and discrimination between peptide bond cleavage (D-AlaD-Ala)



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