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

作者：Cook Paul D; Carney Amanda E; Holden Hazel M^*

来源：Biochemistry, 2008, 47(40): 10685-10693.

DOI：10.1021/bi801309q

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 01 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 angstrom 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 Co1D, catalyzes a dehydration reaction using the same substrate.

• 出版日期2008-10-7