1RA0

Bacterial cytosine deaminase D314G mutant bound to 5-fluoro-4-(S)-hydroxy-3,4-dihydropyrimidine.

1RA0 の概要

• エントリーDOI: 10.2210/pdb1ra0/pdb
• 関連するPDBエントリー: 1K7O 1R9X 1R9Y 1R9Z 1RA5 1RAK 1RB7
• 分子名称: Cytosine deaminase, FE (III) ION, (4S)-5-FLUORO-4-HYDROXY-3,4-DIHYDROPYRIMIDIN-2(1H)-ONE, ... (4 entities in total)
• 機能のキーワード: cytosine deaminase, alpha-beta barrel, hexamer, conformational change, d314g mutant, hydrolase
• 由来する生物種: Escherichia coli
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 47989.90

構造登録者

Mahan, S.D.,Ireton, G.C.,Stoddard, B.L.,Black, M.E. (登録日: 2003-10-31, 公開日: 2004-10-05, 最終更新日: 2023-08-23)

主引用文献

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

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: 15381761
DOI: 10.1093/protein/gzh074

実験手法

X-RAY DIFFRACTION (1.12 Å)