CAREER:Structure-Function Studies on Choline Oxidase

Georgia State University Research Foundation, Inc., Atlanta GA

Investigators

Abstract

This research program focuses on a detailed investigation into the enzymology of choline oxidase (CHO), a flavin-dependent enzyme belonging to a highly diversified superfamily of flavoenzymes that catalyze the oxidation of alcohols, the Glucose-Methanol-Choline (GMC) oxidoreductase superfamily. CHO catalyzes the four-electron oxidation of choline to glycine betaine through two sequential flavin-linked hydride transfer reactions from choline and the ensuing betaine aldehyde intermediate to molecular oxygen. The specific goals of the research program are to: i) Establish the importance of limited independent substrate motion with respect to catalytic groups, so that the only dynamical movements permitted in the activated enzyme-substrate complex are those that contribute to efficient catalysis. ii) Identify the active site amino acid residue that activates the alcohol substrate by removal of the hydroxyl proton. iii) Establish that oxygen activation for reaction with the reduced flavin is provided by the positively charged headgroup of the enzyme-bound aldehyde intermediate. The experimental approach will combine kinetic and biochemical analyses, substrate deuterium, solvent, and 18O-kinetic isotope effects, as well as mutagenesis and crystallography. The results that will be gathered from this project will significantly advance the knowledge and understanding of the enzymology of CHO and, to a broader extent, of the structural and functional factors that are required for efficient catalysis and oxygen reactivity in flavin-dependent enzymes. Broader Impacts The research project will foster the integration of exciting research with the education of minority students, enabling them to develop skills and knowledge in mechanistic enzymology, biochemistry, protein chemistry, and molecular biology. Knowledge and skills in these disciplines are becoming increasingly indispensable in the post-genome era in order to shorten the existing gap between scientific knowledge at the genomic level, where a wealth of information is already available, and post-genomic level, where knowledge is lagging behind. Knowledge and level of training that will be acquired by students through the achievement of the aims of the project will be not only of significance for the understanding of the enzyme under study, but will be an invaluable asset for the future studies of other enzymes and proteins. This work will be pursued in the context of Dr. Gadda's educational activities. He will continue and expand his outreach contribution in the NSF Center for Workshop in the Chemical Sciences and in an interactive two-way television network seminar series that is broadcast live to colleges in Georgia.