1ra5
From Proteopedia
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'''Bacterial cytosine deaminase D314A mutant bound to 5-fluoro-4-(S)-hydroxyl-3,4-dihydropyrimidine.''' | '''Bacterial cytosine deaminase D314A mutant bound to 5-fluoro-4-(S)-hydroxyl-3,4-dihydropyrimidine.''' | ||
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[[Category: Mahan, S D.]] | [[Category: Mahan, S D.]] | ||
[[Category: Stoddard, B L.]] | [[Category: Stoddard, B L.]] | ||
| - | [[Category: | + | [[Category: Alpha-beta barrel]] |
| - | [[Category: | + | [[Category: Conformational change]] |
| - | [[Category: | + | [[Category: Cytosine deaminase]] |
| - | [[Category: | + | [[Category: D314a mutant]] |
| - | [[Category: | + | [[Category: Hexamer]] |
| - | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sat May 3 07:15:46 2008'' | |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | + | |
Revision as of 04:15, 3 May 2008
Bacterial cytosine deaminase D314A mutant bound to 5-fluoro-4-(S)-hydroxyl-3,4-dihydropyrimidine.
Overview
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.
About this Structure
1RA5 is a Single protein structure of sequence from Escherichia coli. Full crystallographic information is available from OCA.
Reference
Random mutagenesis and selection of Escherichia coli cytosine deaminase for cancer gene therapy., Mahan SD, Ireton GC, Knoeber C, Stoddard BL, Black ME, Protein Eng Des Sel. 2004 Aug;17(8):625-33. Epub 2004 Sep 20. PMID:15381761 Page seeded by OCA on Sat May 3 07:15:46 2008
