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Journal of Bacteriology, November 1998, p. 5792-5795, Vol. 180, No. 21
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

DdlN from Vancomycin-Producing Amycolatopsis orientalis C329.2 Is a VanA Homologue with D-Alanyl-D-Lactate Ligase Activity

C. Gary Marshall and Gerard D. Wright^*

Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada L8N 3Z5

Received 25 June 1998/Accepted 31 August 1998

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.

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^* Corresponding author. Mailing address: Department of Biochemistry, McMaster University, 1200 Main St. W., Hamilton, Ontario, Canada L8N 3Z5. Phone: (905) 525-9140, ext. 22943. Fax: (905) 522-9033. E-mail: wrightge{at}fhs.csu.mcmaster.ca.

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Journal of Bacteriology, November 1998, p. 5792-5795, Vol. 180, No. 21
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

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