2jbv
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CRYSTAL STRUCTURE OF CHOLINE OXIDASE REVEALS INSIGHTS INTO THE CATALYTIC MECHANISM
OverviewOverview
Choline oxidase catalyzes the oxidation of choline to glycine betaine, a, compatible solute that accumulates in pathogenic bacteria and plants so, they can withstand osmotic and temperature stresses. The crystal structure, of choline oxidase was determined and refined to a resolution of 1.86 A, with data collected at 100 K using synchrotron X-ray radiation. The, structure reveals a covalent linkage between His99 Nepsilon2 and FAD C8M, atoms, and a 123 A3 solvent-excluded cavity adjacent to the re face of the, flavin. A hypothetical model for choline docked into the cavity suggests, that several aromatic residues and Glu312 may orient the cationic, substrate for efficient catalysis. The role of the negative charge on, Glu312 was investigated by engineering variant enzymes in which Glu312 was, replaced with alanine, glutamine, or aspartate. The Glu312Ala enzyme was, inactive. The Glu312Gln enzyme exhibited a Kd value for choline at least, 500 times larger than that of the wild-type enzyme. The Glu312Asp enzyme, had a kcat/KO2 value similar to that of the wild-type enzyme but kcat and, kcat/Km values that were 230 and 35 times lower, respectively, than in the, wild-type enzyme. These data are consistent with the spatial location of, the negative charge on residue 312 being important for the oxidation of, the alcohol substrate. Solvent viscosity and substrate kinetic isotope, effects suggest the presence of an internal equilibrium in the Glu312Asp, enzyme prior to the hydride transfer reaction. Altogether, the, crystallographic and mechanistic data suggest that Glu312 is important for, binding and positioning of the substrate in the active site of choline, oxidase.
About this StructureAbout this Structure
2JBV is a Single protein structure of sequence from Arthrobacter globiformis with , and as ligands. Active as Oxidoreductase, with EC number 1.1.3.17 Known structural/functional Sites: , , , , , , and . Full crystallographic information is available from OCA.
ReferenceReference
Role of Glu312 in Binding and Positioning of the Substrate for the Hydride Transfer Reaction in Choline Oxidase(,)., Quaye O, Lountos GT, Fan F, Orville AM, Gadda G, Biochemistry. 2007 Dec 12;. PMID:18072756
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