3B8X

Crystal structure of GDP-4-keto-6-deoxymannose-3-dehydratase (ColD) H188N mutant with bound GDP-perosamine

Summary for 3B8X

• Entry DOI: 10.2210/pdb3b8x/pdb
• Related: 2GMU 2R0T
• Descriptor: Pyridoxamine 5-phosphate-dependent dehydrase, SODIUM ION, [(2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl]methyl (2R,3S,4S,5S,6R)-3,4-dihydroxy-5-[({3-hydroxy-2-methyl-5-[(phosphonooxy)methyl]pyridin-4-yl}methyl)amino]-6-methyltetrahydro-2H-pyran-2-yl dihydrogen diphosphate, ... (5 entities in total)
• Functional Keywords: aspartate aminotransferase, colitose, perosamine, o-antigen, plp, pyridoxal phosphate, transferase
• Biological source: Escherichia coli
• Total number of polymer chains: 2
• Total formula weight: 90665.87

Authors

Cook, P.D.,Holden, H.M. (deposition date: 2007-11-02, release date: 2007-11-27, Last modification date: 2023-08-30)

Primary citation

Cook, P.D.,Holden, H.M.
GDP-4-Keto-6-deoxy-D-mannose 3-Dehydratase, Accommodating a Sugar Substrate in the Active Site.
J.Biol.Chem., 283:4295-4303, 2008

PubMed 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 pyridoxal 5'-phosphate-dependent, but unlike most of these proteins, the conserved lysine residue that 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 1.7A 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-dideoxymannose using GDP-perosamine instead of GDP-4-keto-6-deoxymannose as a substrate.

PubMed: 18045869
DOI: 10.1074/jbc.M708893200

Experimental method

X-RAY DIFFRACTION (1.7 Å)