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PEDS Advance Access published online on May 10, 2004
Protein Engineering Design and Selection, doi:10.1093/protein/gzh041
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Received January 21, 2004
Revised April 29, 2004
Accepted April 30, 2004

Article

Library screening studies to investigate substrate specificity in the reaction catalyzed by cholesterol oxidase

J. Xiang 1 N. S. Sampson 1*

1 Department of Chemistry, State University of New York, Stony Brook, NY 11794-3400, USA

* To whom correspondence should be addressed. E-mail: nicole.sampson{at}stonybrook.edu.


  Abstract

We tested whether it is possible to alter the substrate specificity of cholesterol oxidase for similarly sized sterols, i.e., cholesterol, {beta}-sitosterol, and stigmasterol. Using existing X-ray crystal structures, we made a model of the predicted Michaelis complex of cholesterol and cholesterol oxidase. Based on this model, we identified 5 residues that are in direct contact with the steroid tail, Met58, Leu82, Val85, Met365, and Phe433. We prepared 7 mutant libraries that contained the codon NYS (N = A, C, G, T; Y = C, Y; S = C, G) at one, two, or three of the targeted positions by cassette mutagenesis. The libraries were screened for catalytic activity against 3 different sterols under kcat*/Km* conditions with 25 mol% sterol/DOPC unilamellar vesicles. The results of our screens suggest that specific packing interactions are not realized in the transition state of binding and that loss of active site water may be the predominant source of binding energy.

Keywords: selection, library, mutagenesis, sterol, substrate recognition


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