GDP-perosamine synthase: Structural analysis and production of a novel trideoxysugar

Perosarnine 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 0 1 (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) over circle 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 (x-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.