Accommodation of GDP-Linked Sugars in the Active Site of GDP-Perosamine Synthase
- UW
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 {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{sub m} for the unnatural substrate was unperturbed relative to that of the natural substrate, but the k{sub 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.
- Research Organization:
- Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
- Sponsoring Organization:
- USDOE
- OSTI ID:
- 1006938
- Journal Information:
- Biochemistry-US, Vol. 47, Issue (40) ; 2008; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- ENGLISH
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