Accommodation of GDP-linked sugars in the active site of GDP-perosamine synthase

Biochemistry. 2008 Oct 7;47(40):10685-93. doi: 10.1021/bi801309q. Epub 2008 Sep 17.


Perosamine (4-amino-4,6-dideoxy- d-mannose), or its N-acetylated form, is one of several dideoxy sugars found in the O-antigens of such infamous Gram-negative bacteria as Vibrio cholerae O1 and Escherichia coli O157:H7. It is added to the bacterial O-antigen via a nucleotide-linked version, namely GDP-perosamine. Three enzymes are required for the biosynthesis of GDP-perosamine starting from mannose 1-phosphate. The focus of this investigation is GDP-perosamine synthase from Caulobacter crescentus, which catalyzes the final step in GDP-perosamine synthesis, the conversion of GDP-4-keto-6-deoxymannose to GDP-perosamine. The enzyme is PLP-dependent and belongs to the aspartate aminotransferase superfamily. It contains the typically conserved active site lysine residue, which forms a Schiff base with the PLP cofactor. Two crystal structures were determined for this investigation: a site-directed mutant protein (K186A) complexed with GDP-perosamine and the wild-type enzyme complexed with an unnatural ligand, GDP-3-deoxyperosamine. These structures, determined to 1.6 and 1.7 A resolution, respectively, revealed the manner in which products, and presumably substrates, are accommodated within the active site pocket of GDP-perosamine synthase. Additional kinetic analyses using both the natural and unnatural substrates revealed that the K m for the unnatural substrate was unperturbed relative to that of the natural substrate, but the k cat was lowered by a factor of approximately 200. Taken together, these studies shed light on why GDP-perosamine synthase functions as an aminotransferase whereas another very similar PLP-dependent enzyme, GDP-4-keto-6-deoxy- d-mannose 3-dehydratase or ColD, catalyzes a dehydration reaction using the same substrate.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Carbohydrate Epimerases / chemistry*
  • Carbohydrate Epimerases / genetics
  • Carbohydrate Epimerases / metabolism
  • Catalytic Domain / genetics
  • Caulobacter crescentus / enzymology
  • Crystallography, X-Ray
  • Guanosine Diphosphate Sugars / chemistry*
  • Guanosine Diphosphate Sugars / metabolism
  • Kinetics
  • Mannose / analogs & derivatives
  • Mannose / chemistry
  • Mannose / metabolism
  • Models, Molecular
  • Molecular Structure
  • Mutagenesis, Site-Directed
  • Protein Binding
  • Protein Structure, Secondary
  • Substrate Specificity
  • Transaminases / chemistry*
  • Transaminases / genetics
  • Transaminases / metabolism


  • Bacterial Proteins
  • Guanosine Diphosphate Sugars
  • 4-amino-4,6-dideoxy-D-mannose
  • Transaminases
  • Carbohydrate Epimerases
  • perosamine synthetase
  • Mannose

Associated data

  • PDB/3DR4
  • PDB/3DR7