Journal
BIOCHEMISTRY
Volume 47, Issue 1, Pages 243-256Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi7017943
Keywords
-
Categories
Funding
- NCRR NIH HHS [2 P41 RR012408] Funding Source: Medline
Ask authors/readers for more resources
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 angstrom with data collected at 100 K using synchrotron X-ray radiation. The structure reveals a covalent linkage between His99 N epsilon 2 and FAD C8M atoms, and a 123 angstrom(3) 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 K-d value for choline at least 500 times larger than that of the wild-type enzyme. The Glu312Asp enzyme had a k(cat)/k(O2) value similar to that of the wild-type enzyme but k(cat) and k(cat)/k(m) 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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available