title: The UDP-glucose dehydrogenase of Escherichia coli K-12 displays substrate inhibition by NAD that is relieved by nucleotide triphosphates.
abstract: UDP-glucose dehydrogenase (Ugd) generates UDP-glucuronic acid, an important precursor for the production of many hexuronic acid-containing bacterial surface glycostructures. In Escherichia coli K-12, Ugd is important for biosynthesis of the environmentally regulated exopolysaccharide known as colanic acid, whereas in other E. coli isolates, the same enzyme is required for production of the constitutive group 1 capsular polysaccharides, which act as virulence determinants. Recent studies have implicated tyrosine phosphorylation in the activation of Ugd from E. coli K-12, although it is not known if this is a feature shared by bacterial Ugd proteins. The activities of Ugd from E. coli K-12 and from the group 1 capsule prototype (serotype K30) were compared. Surprisingly, for both enzymes, site-directed Tyr → Phe mutants affecting the previously proposed phosphorylation site retained similar kinetic properties to the wild-type protein. Purified Ugd from E. coli K-12 had significant levels of NAD substrate inhibition, which could be alleviated by the addition of ATP and several other nucleotide triphosphates. Mutations in a previously identified UDP-glucuronic acid allosteric binding site decreased the binding affinity of the nucleotide triphosphate. Ugd from E. coli serotype K30 was not inhibited by NAD, but its activity still increased in the presence of ATP.
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
authors.forename: Iain L
authors.initials: IL
authors.lastname: Mainprize
authors.forename: Jordan D
authors.initials: JD
authors.lastname: Bean
authors.forename: Catrien
authors.initials: C
authors.lastname: Bouwman
authors.forename: Matthew S
authors.initials: MS
authors.lastname: Kimber
authors.forename: Chris
authors.initials: C
authors.lastname: Whitfield
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D048168:Escherichia coli K12 / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009243:NAD / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011134:Polysaccharides / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D014533:Uridine Diphosphate Glucose Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D037521:Virulence Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D011134:Polysaccharides; D037521:Virulence Factors; D009243:NAD; D000255:Adenosine Triphosphate; C004275:colanic acid; D014533:Uridine Diphosphate Glucose Dehydrogenase
keywords: Bacterial Metabolism; Carbohydrate Biosynthesis; Carbohydrate Glycoconjugate; Enzymes; Escherichia coli; Glycobiology