title: DdlN from vancomycin-producing Amycolatopsis orientalis C329.2 is a VanA homologue with D-alanyl-D-lactate ligase activity.
abstract: Vancomycin-resistant enterococci acquire high-level resistance to glycopeptide antibiotics through the synthesis of peptidoglycan terminating in D-alanyl-D-lactate. A key enzyme in this process is a D-alanyl-D-alanine ligase homologue, VanA or VanB, which preferentially catalyzes the synthesis of the depsipeptide D-alanyl-D-lactate. We report the overexpression, purification, and enzymatic characterization of DdlN, a VanA and VanB homologue encoded by a gene of the vancomycin-producing organism Amycolatopsis orientalis C329.2. Evaluation of kinetic parameters for the synthesis of peptides and depsipeptides revealed a close relationship between VanA and DdlN in that depsipeptide formation was kinetically preferred at physiologic pH; however, the DdlN enzyme demonstrated a narrower substrate specificity and commensurately increased affinity for D-lactate in the C-terminal position over VanA. The results of these functional experiments also reinforce the results of previous studies that demonstrated that glycopeptide resistance enzymes from glycopeptide-producing bacteria are potential sources of resistance enzymes in clinically relevant bacteria.
authors.affiliation: Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada L8N 3Z5.
authors.forename: C G
authors.initials: CG
authors.lastname: Marshall
authors.forename: G D
authors.initials: GD
authors.lastname: Wright
mesh_terms: D000191:Actinomycetaceae / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D019729:Carbon-Oxygen Ligases / Q000235:genetics / Q000378:metabolism*; D004151:Dipeptides / Q000096:biosynthesis; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D010452:Peptide Biosynthesis; D013379:Substrate Specificity; D014640:Vancomycin
chemical_list: D001426:Bacterial Proteins; D004151:Dipeptides; C071437:VanA ligase, Bacteria; D014640:Vancomycin; D019729:Carbon-Oxygen Ligases