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Date of Award


Degree Type

Closed Dissertation

Degree Name

Doctor of Philosophy (PhD)



First Advisor

Giovanni Gadda - Chair

Second Advisor

Phang C. Tai

Third Advisor

Chung-Dar Lu

Fourth Advisor

Dale E. Edmondson

Fifth Advisor

Dabney W. Dixon


Choline oxidase catalyzes the four-electron oxidation of choline to glycine betaine, a limited number of compounds that accumulate to high levels in cytoplasm to prevent dehydration and plasmolysis in adverse hyperosmotic environments. With this respect, the study of choline oxidase has potential for the development of therapeutic agents that inhibit the biosynthesis of glycine betaine, thereby rendering pathogenic bacteria susceptible to either conventional treatments or the immune system. In this study, the highly GC rich codA gene encoding for choline oxidase was cloned, expressed. The resulting enzyme was purified to high levels, allowing for detailed biochemical, mechanistic and structural characterizations. A chemical mechanism for the reaction catalyzed by choline oxidase was established by using kinetic isotope effects and viscosity effects as probes, in which the choline hydroxyl proton is not in flight in the transition state for CH bond cleavage. Furthermore, these experiments indicated that chemical steps of flavin reduction by choline and betaine aldehyde are rate limiting for the overall turnover of the enzyme. Further mechanistic characterization clearly suggested a hydride transfer mechanism that is fully quantum mechanical. The structure of choline oxidase was resolved at 1.86 Å resolution in collaboration with the group of Dr. Allen O. Orville, at the Georgia Institute of Technology, providing a structural framework that is consistent with the mechanistic studies. The results of these studies will be presented and discussed in the context of the Glucose-Methanol-Choline oxidoreductase enzyme superfamily, of which choline oxidase is a member. Previous structural and mechanistic studies of alcohol- and aldehyde-oxidizing enzymes with different cofactors, as well as the biotechnological and biomedical relevance of choline oxidase are presented in Chapter 1. Chapter 3-8 illustrate my studies on choline oxidase, including cloning, expression, purification and preliminary characterizations (Chapter 3), spectroscopic and steady state kinetics (Chapter 4), the determination of the chemical mechanism for alcohol oxidation and the investigation of the involvement of quantum mechanical tunneling (Chapter 5 and 6), the study of aldehyde oxidation (Chapter 7), and the structural determination of choline oxidase by x-ray crystallography (Chapter 8). Chapter 9 presents a general discussion of the data presented.