The position of a key tyrosine in dTDP-4-keto-6-deoxy-D-glucose-5-epimerase (EvaD) alters the substrate profile for this RmlC-like enzyme

Alexandra B Merkel, Louise L Major, James C Errey, Michael D Burkart, Robert A Field, Christopher T Walsh, James H Naismith

Research output: Contribution to journalArticlepeer-review

22 Citations (Scopus)

Abstract

Vancomycin, the last line of defense antibiotic, depends upon the attachment of the carbohydrate vancosamine to an aglycone skeleton for antibacterial activity. Vancomycin is a naturally occurring secondary metabolite that can be produced by bacterial fermentation. To combat emerging resistance, it has been proposed to genetically engineer bacteria to produce analogues of vancomycin. This requires a detailed understanding of the biochemical steps in the synthesis of vancomycin. Here we report the 1.4 Angstrom structure and biochemical characterization of EvaD, an RmlC-like protein that is required for the C-5' epimerization during synthesis of dTDP-epivancosamine. EvaD, although clearly belonging to the RmlC class of enzymes, displays very low activity in the archetypal RmlC reaction ( double epimerization of dTDP-6-deoxy-4-keto-D-glucose at C-3' and C-5'). The high resolution structure of EvaD compared with the structures of authentic RmlC enzymes indicates that a subtle change in the enzyme active site repositions a key catalytic Tyr residue. A mutant designed to re-establish the normal position of the Tyr increases the RmlC-like activity of EvaD.

Original languageEnglish
Pages (from-to)32684-32691
Number of pages8
JournalJournal of Biological Chemistry
Volume279
Issue number31
DOIs
Publication statusPublished - 30 Jul 2004

Keywords

  • Amino Acid Sequence
  • Anti-Bacterial Agents/pharmacology
  • Carbohydrate Epimerases/chemistry
  • Carbohydrate Sequence
  • Carbohydrates
  • Catalysis
  • Circular Dichroism
  • Crystallography, X-Ray
  • Kinetics
  • Mass Spectrometry
  • Models, Chemical
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis
  • Mutagenesis, Site-Directed
  • Protein Conformation
  • Protein Structure, Secondary
  • Salmonella enterica/metabolism
  • Sequence Homology, Amino Acid
  • Structure-Activity Relationship
  • Substrate Specificity
  • Tyrosine/chemistry
  • Vancomycin/chemistry

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