1RA5
Bacterial cytosine deaminase D314A mutant bound to 5-fluoro-4-(S)-hydroxyl-3,4-dihydropyrimidine.
Summary for 1RA5
| Entry DOI | 10.2210/pdb1ra5/pdb |
| Related | 1K7O 1R9X 1R9Y 1R9Z 1RA0 1RAK 1RB7 |
| Descriptor | Cytosine deaminase, FE (III) ION, (4S)-5-FLUORO-4-HYDROXY-3,4-DIHYDROPYRIMIDIN-2(1H)-ONE, ... (5 entities in total) |
| Functional Keywords | cytosine deaminase, alpha-beta barrel, hexamer, conformational change, d314a mutant, hydrolase |
| Biological source | Escherichia coli |
| Total number of polymer chains | 1 |
| Total formula weight | 48096.02 |
| Authors | Mahan, S.D.,Ireton, G.C.,Stoddard, B.L.,Black, M.E. (deposition date: 2003-10-31, release date: 2004-10-05, Last modification date: 2024-04-03) |
| Primary citation | Mahan, S.D.,Ireton, G.C.,Knoeber, C.,Stoddard, B.L.,Black, M.E. Random mutagenesis and selection of Escherichia coli cytosine deaminase for cancer gene therapy. Protein Eng.Des.Sel., 17:625-633, 2004 Cited by PubMed 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-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. PubMed: 15381761DOI: 10.1093/protein/gzh074 PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (1.4 Å) |
Structure validation
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