title: GDP-perosamine synthase: structural analysis and production of a novel trideoxysugar.
abstract: Perosamine or 4-amino-4,6-dideoxy- d-mannose is an unusual sugar found in the O-antigens of some Gram-negative bacteria such as Vibrio cholerae O1 (the causative agent of cholera) or Escherichia coli O157:H7 (the leading cause of food-borne illnesses). It and similar deoxysugars are added to the O-antigens of bacteria via the action of glycosyltransferases that employ nucleotide-linked sugars as their substrates. The focus of this report is GDP-perosamine synthase, a PLP-dependent enzyme that catalyzes the last step in the formation of GDP-perosamine, namely, the amination of the sugar C-4'. Here we describe the three-dimensional structure of the enzyme from Caulobacter crescentus determined to a nominal resolution of 1.8 A and refined to an R-factor of 17.9%. The overall fold of the enzyme places it into the well-characterized aspartate aminotransferase superfamily. Each subunit of the dimeric enzyme contains a seven-stranded mixed beta-sheet, a two-stranded antiparallel beta-sheet, and 12 alpha-helices. Amino acid residues from both subunits form the active sites of the GDP-perosamine synthase dimer. Recently, the structure of another PLP-dependent enzyme, GDP-4-keto-6-deoxy- d-mannose-3-dehydratase (or ColD), was determined in our laboratory, and this enzyme employs the same substrate as GDP-perosamine synthase. Unlike GDP-perosamine synthase, however, ColD functions as a dehydratase that removes the sugar C-3' hydroxyl group. By purifying the ColD product and reacting it with purified GDP-perosamine synthase, we have produced a novel GDP-linked sugar, GDP-4-amino-3,4,6-trideoxy- d-mannose. Details describing the X-ray structural investigation of GDP-perosamine synthase and the enzymatic synthesis of GDP-4-amino-3,4,6-trideoxy- d-mannose are presented.
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Cook
authors.forename: Hazel M
authors.initials: HM
authors.lastname: Holden
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000378:metabolism*; D016935:Caulobacter crescentus / Q000201:enzymology / Q000378:metabolism; D018360:Crystallography, X-Ray; D003837:Deoxy Sugars / Q000737:chemistry / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000737:chemistry / Q000378:metabolism*; D008358:Mannose / Q000031:analogs & derivatives / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D040681:Structural Homology, Protein; D000637:Transaminases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D003837:Deoxy Sugars; D006153:Guanosine Diphosphate; C057033:4-amino-4,6-dideoxy-D-mannose; D000637:Transaminases; D002238:Carbohydrate Epimerases; C061272:perosamine synthetase; D008358:Mannose