PEDS Advance Access originally published online on May 10, 2004
Protein Engineering Design and Selection 2004 17(4):341-348; doi:10.1093/protein/gzh041
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Library screening studies to investigate substrate specificity in the reaction catalyzed by cholesterol oxidase
Department of Chemistry, State University of New York, Stony Brook, NY 11794-3400, USA
1 To whom correspondence should be addressed. E-mail: nicole.sampson{at}stonybrook.edu
We tested whether it is possible to alter the substrate specificity of cholesterol oxidase for similarly sized sterols, i.e. cholesterol, ß-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 five residues that are in direct contact with the steroid tail, Met58, Leu82, Val85, Met365 and Phe433. We prepared seven 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 three 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.
Received January 21, 2004; revised April 29, 2004; accepted April 30, 2004.
Edited by Derek N.Woolfson