D-Ala-D-X ligases: evaluation of D-alanyl phosphate intermediate by MIX, PIX and rapid quench studies.

BACKGROUND: The D-alanyl-D-lactate (D-Ala-D-Lac) ligase is required for synthesis of altered peptidoglycan (PG) termini in the VanA phenotype of vancomycin-resistant enterococci (VRE), and the D-alanyl-D-serine (D-Ala-D-Ser) ligase is required for the VanC phenotype of VRE. Here we have compared these with the Escherichia coli D-Ala-D-Ala ligase DdlB for formation of the enzyme-bound D-alanyl phosphate, D-Ala(1)-PO(3)(2-) (D-Ala(1)-P), intermediate. RESULTS: The VanC2 ligase catalyzes a molecular isotope exchange (MIX) partial reaction, incorporating radioactivity from (14)C-D-Ser into D-Ala-(14)C-D-Ser at a rate of 0.7 min(-1), which approaches kinetic competence for the reversible D-Ala(1)-P formation from the back direction. A positional isotope exchange (PIX) study with the VanC2 and VanA ligases displayed a D-Ala(1)-dependent bridge to nonbridge exchange of the oxygen-18 label of [gamma-(18)O(4)]-ATP at rates of up to 0.6 min(-1); this exchange was completely suppressed by the addition of the second substrate D-Ser or D-Lac, respectively, as the D-Ala(1)-P intermediate was swept in the forward direction. As a third criterion for formation of bound D-Ala(1)-P, we conducted rapid quench studies to detect bursts of ADP formation in the first turnover of DdlB and VanA. With E. coli DdlB, there was a burst amplitude of ADP corresponding to 26-30% of the DdlB active sites, followed by the expected steady-state rate of 620-650 min(-1). For D-Ala-D-Lac and D-Ala-D-Ala synthesis by VanA, we measured a burst of 25-30% or 51% of active enzyme, respectively. CONCLUSIONS: These three approaches support the rapid (more than 1000 min(-1)), reversible formation of the enzyme intermediate D-Ala(1)-P by members of the D-Ala-D-X (where X is Ala, Ser or Lac) ligase superfamily.

Vancomycin resistance in enterococci: reprogramming of the D-ala-D-Ala ligases in bacterial peptidoglycan biosynthesis.

Vancomycin binds to bacterial cell-wall intermediates to achieve its antibiotic effect. Infections of vancomycin-resistant enterococci are, however, becoming an increasing problem; the bacteria are resistant because they synthesize different cell-wall intermediates. The enzymes involved in cell-wall biosynthesis, therefore, are potential targets for combating this resistance. Recent biochemical and crystallographic results are providing mechanistic and structural details about some of these targets.

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 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 beta-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 A 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.

Determinants for differential effects on D-Ala-D-lactate vs D-Ala-D-Ala formation by the VanA ligase from vancomycin-resistant enterococci.

Bacteria with either intrinsic or inducible resistance to vancomycin make peptidoglycan (PG) precursors of lowered affinity for the antibiotic by switching the PG-D-Ala-D-Ala termini that are the antibiotic-binding target to either PG-D-Ala-D-lactate or PG-D-Ala-D-Ser as a consequence of altered specificity of the D-Ala-D-X ligases in the cell wall biosynthetic pathway. The VanA ligase of vancomycin-resistant enterococci, a D-Ala-D-lactate depsipeptide ligase, has the ability to recognize and activate the weak nucleophile D-lactate selectively over D-Ala(2) to capture the D-Ala(1)-OPO(3)(2)(-) intermediate in the ligase active site. To ensure this selectivity in catalysis, VanA largely rejects the protonated (NH(3)(+)) form of D-Ala at subsite 2 (K(M2) of 210 mM at pH 7.5) but not at subsite 1. In contrast, the deprotonated (NH(2)) form of D-Ala (K(M2) of 0.66 mM, k(cat) of 550 min(-)(1)) is a 17-fold better substrate compared to D-lactate (K(M) of 0.69 mM, k(cat) of 32 min(-)(1)). The low concentration of the free amine form of D-Ala at physiological conditions (i.e., 0.1% at pH 7.0) explains the inefficiency of VanA in dipeptide synthesis. Mutational analysis revealed a residue in the putative omega-loop region, Arg242, which is partially responsible for electrostatically repelling the protonated form of D-Ala(2). The VanA enzyme represents a subfamily of D-Ala-D-X ligases in which two key active-site residues (Lys215 and Tyr216) in the active-site omega-loop of the Escherichia coli D-Ala-D-Ala ligase are absent. To look for functional complements in VanA, we have mutated 20 residues and evaluated effects on catalytic efficiency for both D-Ala-D-Ala dipeptide and D-Ala-D-lactate depsipeptide ligation. Mutation of Asp232 caused substantial defects in both dipeptide and depsipeptide ligase activity, suggesting a role in maintaining the loop position. In contrast, the H244A mutation caused an increase in K(M2) for D-lactate but not D-Ala, indicating a differential role for His244 in the recognition of the weaker nucleophile D-lactate. Replacement of the VanA omega-loop by that of VanC2, a D-Ala-D-Ser ligase, eliminated D-Ala-D-lactate activity while improving by 3-fold the catalytic efficacy of D-Ala-D-Ala and D-Ala-D-Ser activity.

Active-site mutants of the VanC2 D-alanyl-D-serine ligase, characteristic of one vancomycin-resistant bacterial phenotype, revert towards wild-type D-alanyl-D-alanine ligases.

BACKGROUND: The rising number of vancomycin-resistant enterococci (VREs) is a major concern to modern medicine because vancomycin is currently the 'last resort' drug for life-threatening infections. The D-alanyl-D-X ligases (where X is an hydroxy or amino acid) of bacteria catalyze a critical step in bacterial cell-wall peptidoglycan assembly. In bacteria that produce glycopeptide antibiotics and in opportunistic pathogens, including VREs, D-, D-ligases serve as switches that confer antibiotic resistance on the bacteria themselves. Peptidoglycans in vancomycin-sensitive bacteria end in D-alanyl-D-alanine, whereas in vancomycin-resistant bacteria they end in D-alanyl-D-lactate or D-alanyl-D-serine. RESULTS: We demonstrate that the selective utilization of D-serine by the Enterococcus casseliflavus VanC2 ligase can be altered by mutagenesis of one of two residues identified by homology to the X-ray structure of the Escherichia coli D-alanyl-Dalanine ligase (DdlB). The Arg322-->Met (R322M) and Phe250-->Tyr (F250Y) ligase mutants show a 36-44-fold decrease in the use of D-serine, as well as broadened specificity for utilization of other D-amino acids in place of D-serine. The F250Y R322M double mutant is effectively disabled as a D-alanyl-D-serine ligase and retains 10% of the catalytic activity of wild-type D-alanyl-D-alanine ligases, reflecting a 6,000-fold switch to the D-alanyl-D-alanine peptide. Correspondingly, the Leu282-->Arg mutant of the wild-type E. coli DdlB produced a 560-fold switch towards D-alanyl-D-serine formation. CONCLUSIONS: Single-residue changes in the active-site regions of D-, D-ligases can cause substantial changes in recognition and activation of hydroxy or amino acids that have consequences for glycopeptide antibiotic efficacy. The observations reported here should provide an approach for combatting antibiotic-resistant bacteria.