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B6db references: 18045869

type Journal Article
authors Cook, P.D.; Holden, H.M.
title GDP-4-Keto-6-deoxy-D-mannose-3-dehydratase: Accommodating a sugar substrate in the active site
journal J Biol Chem
Activity cold
Family cold
sel unselected
ui 18045869
year (2008)
volume 283
pages 4295-303
 
abstract Colitose is a dideoxysugar found in the O-antigen of the lipopolysaccharide that coats the outer membrane of some Gram-negative bacteria. Four enzymes are required for its production starting from D-mannose-1-phosphate and GTP. The focus of this investigation is GDP-4-keto-6-deoxy-D-mannose-3-dehydratase or ColD, which catalyzes the removal of the C3'-hydroxyl group from GDP-4-keto-6-deoxymannose. The enzyme is PLP-dependent, but unlike most of these proteins, the conserved lysine residue which covalently holds the cofactor in the active site is replaced with a histidine residue. Here we describe the three-dimensional structure of ColD determined to 1.7 A resolution whereby the active site histidine has been replaced with an asparagine residue. For this investigation, crystals of the site-directed mutant protein were grown in the presence of GDP-4-amino-4,6-dideoxy-D-mannose (GDP-perosamine). The electron density map clearly reveals the presence of the sugar analog trapped in the active site as an external aldimine. The active site is positioned between the two subunits of the dimer. Whereas the pyrophosphoryl groups of the ligand are anchored to the protein via Arg 219 and Arg 331, the hydroxyl groups of the hexose only lie within hydrogen bonding distance to ordered water molecules. Interestingly, the hexose moiety of the ligand adopts a boat rather than the typically observed chair conformation. Activity assays demonstrate that this mutant protein cannot catalyze the dehydration step. Additionally, we report data revealing that wild-type ColD is able to catalyze the production of GDP-4-keto-3,6-dideoxy-mannose using GDP-perosamine instead of GDP-4-keto-6-deoxymannose as a substrate.
last changed 2008/04/01 15:44

B6db references