PEDS Advance Access originally published online on September 20, 2004
Protein Engineering Design and Selection 2004 17(8):625-633; doi:10.1093/protein/gzh074
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Random mutagenesis and selection of Escherichia coli cytosine deaminase for cancer gene therapy
1Department of Pharmaceutical Sciences and the School of Molecular Biosciences, PO Box 646534, Washington State University, Pullman, WA 99164-6534 and 2Fred Hutchinson Cancer Research Center, and the Graduate Program in Molecular and Cell Biology, University of Washington, 1100 Fairview Avenue North A3-023, Seattle, WA 98109, USA
3 To whom correspondence should be addressed. E-mail: blackm{at}mail.wsu.edu
Cytosine deaminase (CD) is currently being used as a suicide gene for cancer gene therapy. The premise of this therapy is the preferential deamination of 5-fluorocytosine (5FC) to 5-fluorouracil by cancer cells expressing cytosine deaminase. However, a lack of efficient gene transfer to tumors combined with inefficient 5FC turnover currently limits the clinical applications of this gene therapy approach. We have used random mutagenesis to create novel bacterial cytosine deaminases that demonstrate an increased preference for 5FC over cytosine. Among the 15 mutants isolated, one conferred sensitivity to Escherichia coli in a negative selection system at a concentration of 5FC that was 10-fold lower than a sublethal dose for wild-type CD. Evaluation of individual substitutions found in this double mutant (Q102R, D314G) demonstrated that the substitution at residue D314 was solely responsible for the observed increase in sensitivity to 5FC. Additional mutagenesis at D314 resulted in the identification of two more substitutions with the ability to confer enhanced 5FC sensitivity to E.coli. Structure determinations of the three CD variants in the presence and absence of a transition state 5FC analogue provide insights to the determinants of substrate binding specificity at the 5' position of the pyrimidine ring. CD mutant D314A is a promising candidate for further gene therapy studies.
Received June 10, 2004; revised August 31, 2004; accepted September 6, 2004.
Edited by Dan Tawfik
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