Enzymes of vancomycin resistance: the structure of D-alanine-D-lactate ligase of naturally resistant Leuconostoc mesenteroides

Background: The bacterial cell wall and the enzymes that synthesize it are targets of glycopeptide antibiotics (vancomycins and teicoplanins) and beta -lactams (penicillins and cephalosporins). Biosynthesis of cell wall peptid oglycan requires a crosslinking of peptidyl moieties on adjacent glycan str ands. The D-alanine-D-alanine transpeptidase, which catalyzes this crosslin king, is the target of beta-lactam antibiotics. Glycopeptides, in contrast, do not inhibit an enzyme, but bind directly to D-alanine-D-alanine and pre vent subsequent crosslinking by the transpeptidase. Clinical resistance to vancomycin in enterococcal pathogens has been traced to altered ligases pro ducing D-alanine-D-lactate rather than D-alanine-D-alanine. Results: The structure of a D-alanine-D-lactate ligase has been determined by multiple anomalous dispersion (MAD) phasing to 2.4 Angstrom resolution. Go-crystallization of the Leuconostoc mesenteroides LmDdl2 ligase with ATP and a di-D-methylphosphinate produced ADP and a phosphinophosphate analog o f the reaction intermediate of cell wall peptidoglycan biosynthesis. Compar ison of this D-alanine-D-lactate ligase with the known structure of DdlB D- alanine-D-alanine ligase, a wild-type enzyme that does not provide vancomyc in resistance, reveals alterations in the size and hydrophobicity of the si te for D-lactate binding (subsite 2). A decrease was noted in the ability o f the ligase to hydrogen bond a substrate molecule entering subsite 2. Conclusions: Structural differences at subsite 2 of the D-alanine-D-lactate ligase help explain a substrate specificity shift (D-alanine to D-lactate) leading to remodeled cell wall peptidoglycan and vancomycin resistance in Gram-positive pathogens.