Web Release Date: December 12,
Role of Glu312 in Binding and Positioning of the Substrate for the Hydride
Transfer Reaction in Choline Oxidase
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Departments of Chemistry and Biology and The Center for Biotechnology and Drug Design, Georgia State University, Atlanta, Georgia 30302-4098, and School of Chemistry and Biochemistry and Parker Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332-0400
Received August 31, 2007
Revised Manuscript Received November 2, 2007
Abstract:
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 Å with data
collected at 100 K using synchrotron X-ray radiation. The structure reveals a covalent linkage between
His99 N
2 and FAD C8M atoms, and a 123 Å3 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.
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