Date of Award

Spring 2004

Document Type

Thesis

Department

Life & Environmental Sciences

First Advisor

Ron Wilde

Second Advisor

Marilyn Schendel

Third Advisor

Sam Alvey

Abstract

Cholesterol oxidase, a bifunctional flavoenzyme facilitates the oxidation and isomerization of cholesterol. Wild type cholesterol oxidase has been crystallized, and its structure has been resolved to 0.95 A. Wild type cholesterol oxidase turns over cholesterol with such extreme efficiency that it is very difficult to crystallize the substrate-enzyme complex. Cholesterol oxidase H447E/E361Q double mutant, which binds cholesterol, but is unable to completely turn it over, was expressed, purified, and crystallized to obtain a crystal structure. Once solved, the structure of the cholesterol oxidase double mutant could then be used as a background structure for the crystallization of the substrate-enzyme complex. Cholesterol oxidase H447E/E361Q double mutant, which binds cholesterol, but is unable to completely turn it over, was expressed, purified, and crystallized to obtain a crystal structure. Once solved, the structure of the cholesterol oxidase double mutant could then be used as a background structure for the crystallization of the substrate-enzyme complex. In an attempt to obtain a control for future experimentation on the substrate-enzyme complex, x-ray crystallographic methods were utilized. These methods yielded high-resolution data to 1.5 A.

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