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PEDS Advance Access published online on September 20, 2004
Protein Engineering Design and Selection, doi:10.1093/protein/gzh074
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Received June 10, 2004
Revised August 31, 2004
Accepted September 6, 2004

Article

Random mutagenesis and selection of E. coli cytosine deaminase for cancer gene therapy

Sheri D. Mahan 1, Greg C. Ireton 2, Catherine Knoeber 1, Barry L. Stoddard 2, and Margaret E. Black 1*

1 Department of Pharmaceutical Sciences and the School of Molecular Biosciences, P. O. Box 646534, Washington State University, Pullman, WA 99164-6534
2 Fred Hutchinson Cancer Research Center, and the Graduate Program in Molecular and Cell Biology, University of Washington, 1100 Fairview Ave. N. A3-023, Seattle, WA 98109

* To whom correspondence should be addressed. E-mail: blackm{at}mail.wsu.edu.


  Abstract

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-flurorouracil 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 E. coli in a negative selection system at a concentration 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. Cytosine deaminase mutant D314A is a promising candidate for further gene therapy studies.

Keywords: cytosine deaminase; gene therapy; 5-fluorocytosine; mutagenesis.
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