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It has been reported that the frequency of a direct crosslink between two diaminopimelate (A2pm) residues on adjacent PG strands rises from 2% in exponential phase to 13% in stationary phase (27). Although the substrate strands for this unusual crosslink have yet not been identified, if they are the normal pentapeptide strands, this crosslink would release the D-Ala-D-Ala dipeptide. The E.coli A2pm-A2pm transpeptidase has not been identified, but it has been suggested that the L, D-dipeptidylcarboxypeptidase cleaving the muramylpeptidyl-L-A2pm-D-Ala-D-Ala at the L-A2pm-D-Ala peptide bond releasing D-Ala-D-Ala previously described in E.coli (28) could be the long-sought A2pm-A2pm transpeptidase (15). The purpose of the reprogramming of the crosslinks in stationary phase may be driven by the transshipment of the released D, D-dipeptide from periplasm back into cytoplasm to power the cell in starvation mode. Although the cell is catabolizing the cell wall, it cannot decrease the net crosslinking or the mechanical strength will be insufficient to withstand osmotic pressure for lysis, hence the need to switch to A2pm-A2pm linkages.

VanX, a Dipeptidase for All Seasons? The three examples noted in this paper reveal distinct niches for the zinc-dependent D-Ala-D-Ala dipeptidase. It may have arisen in the Gram-positive glycopeptide antibiotic producers at the same time as the ability to biosynthesize these antibiotics, providing selective immunity to the bacteria that could both make the antibiotics and reprogram their cell walls to lower the target affinity. Other Gram-positive bacteria in the soil such as lactobacilli, leuconostoc, and pediococci are intrinsically resistant to vancomycin and were examined (2932) to have also chosen the D-Ala-D-lactate route. In recent times, the opportunistic enterococci have imported the vanHAX gene operon on transposons and plasmids to gain survival advantage via antibiotic resistance in hospital environments that have seen an order of magnitude increase in the therapeutic use of vancomycin in the past 15 yr (33). The Gram-negative E.coli is not challenged by the impermeable glycopeptide antibiotics and has its own version of VanX, but not VanH or VanA. DdpX is a potentially lethal enzyme, because it removes the necessary metabolite D-Ala-D-Ala during peptidoglycan synthesis and is turned on only in the extreme challenge of stationary phase when starvation threatens and the D-Ala-D-Ala termini of uncrosslinked peptidoglycan strands are retrieved from the periplasm and burned as a metabolic fuel. As additional bacterial genomes are sequenced, more VanX protein homologs are likely to be discovered. Indeed, in the Gram-negative Synechocystis sp PCC6803, a VanX homolog (16% similarity with EntVanX) possessing kinetic parameters similar to DdpX was detected, but notably it hydrolyzes both L, D- and D, D-dipeptides similarly. It is thus proposed to play a role in scavenging both L, D- and D, D-dipeptide products of cell-wall degradation pathways (15). In the Gram-positive pathogen Mycobacterium tuberculosis, the VanX homolog (21% similarity with EntVanX) possesses all the requirements necessary for dipeptide recognition and catalysis but presents an apparent signal sequence and membrane lipoprotein attachment site, suggesting that MtuVanX might reside in the membrane.

We are grateful to Abbott Laboratories for providing the coordinates of the EntVanX crystal structure. We thank members of the Walsh laboratory for helpful and insightful discussions. I.A.D.L. acknowledges the Medical Research Council of Canada for Postdoctoral Fellowship supports. This research was supported in part by National Institutes of Health Grants GM21643 and by funds from Abbott Laboratories.

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