Toward Solving the Structure of Cholesterol Oxidase H447E/E361Q Double Mutant
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Authors
Krause, Larryn
Date of Issue
2004-04-01
Type
thesis
Language
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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.