title: 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.
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 A 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.
authors.affiliation: Centre for Biomolecular Sciences, The University, St. Andrews, Scotland, KY16 9ST, United Kingdom.
authors.forename: Alexandra B
authors.initials: AB
authors.lastname: Merkel
authors.forename: Louise L
authors.initials: LL
authors.lastname: Major
authors.forename: James C
authors.initials: JC
authors.lastname: Errey
authors.forename: Michael D
authors.initials: MD
authors.lastname: Burkart
authors.forename: Robert A
authors.initials: RA
authors.lastname: Field
authors.forename: Christopher T
authors.initials: CT
authors.lastname: Walsh
authors.forename: James H
authors.initials: JH
authors.lastname: Naismith
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002238:Carbohydrate Epimerases / Q000737:chemistry*; D002240:Carbohydrate Sequence; D002241:Carbohydrates; D002384:Catalysis; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D007700:Kinetics; D013058:Mass Spectrometry; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D019779:Salmonella enterica / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014443:Tyrosine / Q000737:chemistry*; D014640:Vancomycin / Q000737:chemistry / Q000494:pharmacology*; D001426:Bacterial Proteins; D002237:Carbohydrate Dehydrogenases
chemical_list: D000900:Anti-Bacterial Agents; D002238:Carbohydrate Epimerases; D002241:Carbohydrates; D014443:Tyrosine; D014640:Vancomycin; C470843:dTDP-4-keto-6-deoxyglucose-5-epimerase; C020092:dTDP-4-ketorhamnose 3,5-epimerase; D001426:Bacterial Proteins; D002237:Carbohydrate Dehydrogenases