Alanine-scanning mutagenesis reveals a cytosine deaminase mutant with altered substrate preference

Biochemistry. 2004 Jul 20;43(28):8957-64. doi: 10.1021/bi049720z.

Abstract

Suicide gene therapy of cancer is a method whereby cancerous tumors can be selectively eradicated while sparing damage to normal tissue. This is accomplished by delivering a gene, encoding an enzyme capable of specifically converting a nontoxic prodrug into a cytotoxin, to cancer cells followed by prodrug administration. The Escherichia coli gene, codA, encodes cytosine deaminase and is introduced into cancer cells followed by administration of the prodrug 5-fluorocytosine (5-FC). Cytosine deaminase converts 5-FC into cytotoxic 5-fluorouracil, which leads to tumor-cell eradication. One limitation of this enzyme/prodrug combination is that 5-FC is a poor substrate for bacterial cytosine deaminase. The crystal structure of bacterial cytosine deaminase (bCD) reveals that a loop structure in the active site pocket of wild-type bCD comprising residues 310-320 undergoes a conformational change upon cytosine binding, making several contacts to the pyrimidine ring. Alanine-scanning mutagenesis was used to investigate the structure-function relationship of amino acid residues within this region, especially with regard to substrate specificity. Using an E. coli genetic complementation system, seven active mutants were identified (F310A, G311A, H312A, D314A, V315A, F316A, and P318A). Further characterization of these mutants reveals that mutant F316A is 14-fold more efficient than the wild-type at deaminating cytosine to uracil. The mutant D314A enzyme demonstrates a dramatic decrease in cytosine activity (17-fold) as well as a slight increase in activity toward 5-FC (2-fold), indicating that mutant D314A prefers the prodrug over cytosine by almost 20-fold, suggesting that it may be a superior suicide gene.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alanine
  • Amino Acid Sequence
  • Antineoplastic Agents
  • Binding Sites
  • Crystallization
  • Cytosine / metabolism
  • Cytosine Deaminase / genetics*
  • Cytosine Deaminase / metabolism
  • Escherichia coli / genetics
  • Escherichia coli Proteins
  • Flucytosine / metabolism
  • Genes, Transgenic, Suicide*
  • Genetic Complementation Test
  • Mutagenesis, Site-Directed*
  • Prodrugs / metabolism
  • Structure-Activity Relationship
  • Substrate Specificity / genetics

Substances

  • Antineoplastic Agents
  • Escherichia coli Proteins
  • Prodrugs
  • Cytosine
  • Flucytosine
  • Cytosine Deaminase
  • codA protein, E coli
  • Alanine