title: D-Ala-D-X ligases: evaluation of D-alanyl phosphate intermediate by MIX, PIX and rapid quench studies.
abstract: 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.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: V L
authors.initials: VL
authors.lastname: Healy
authors.forename: L S
authors.initials: LS
authors.lastname: Mullins
authors.forename: X
authors.initials: X
authors.lastname: Li
authors.forename: S E
authors.initials: SE
authors.lastname: Hall
authors.forename: F M
authors.initials: FM
authors.lastname: Raushel
authors.forename: C T
authors.initials: CT
authors.lastname: Walsh
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019729:Carbon-Oxygen Ligases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D016983:Enterococcus / Q000201:enzymology*; D004791:Enzyme Inhibitors; D007553:Isotope Labeling; D007700:Kinetics; D015394:Molecular Structure; D010453:Peptide Synthases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010457:Peptidoglycan / Q000187:drug effects / Q000378:metabolism; D013379:Substrate Specificity; D020713:Vancomycin Resistance
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D010457:Peptidoglycan; C071437:VanA ligase, Bacteria; D000255:Adenosine Triphosphate; D019729:Carbon-Oxygen Ligases; D010453:Peptide Synthases; C108395:VanC protein, Enterococcus; C019638:D-alanylalanine synthetase