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

AbstractBackground: The bacterial cell wall and the enzymes that synthesize it are targets of glycopeptide antibiotics (vancomycins and teicoplanins) and β-lactams (penicillins and cephalosporins). Biosynthesis of cell wall peptidoglycan requires a crosslinking of peptidyl moieties on adjacent glycan strands. The d-alanine–d-alanine transpeptidase, which catalyzes this crosslinking, is the target of β-lactam antibiotics. Glycopeptides, in contrast, do not inhibit an enzyme, but bind directly to d-alanine–d-alanine and prevent subsequent crosslinking by the transpeptidase. Clinical resistance to vancomycin in enterococcal pathogens has been traced to altered ligases producing 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 Å resolution. Co-crystallization of the Leuconostoc mesenteroides LmDdl2 ligase with ATP and a di-d-methylphosphinate produced ADP and a phosphinophosphate analog of the reaction intermediate of cell wall peptidoglycan biosynthesis. Comparison 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 vancomycin resistance, reveals alterations in the size and hydrophobicity of the site for d-lactate binding (subsite 2). A decrease was noted in the ability of 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