1R9X

Bacterial cytosine deaminase D314G mutant.


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.58 Å
  • R-Value Free: 0.184 
  • R-Value Work: 0.164 
  • R-Value Observed: 0.164 

wwPDB Validation   3D Report Full Report


This is version 1.4 of the entry. See complete history


Literature

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

Mahan, S.D.Ireton, G.C.Knoeber, C.Stoddard, B.L.Black, M.E.

(2004) Protein Eng Des Sel 17: 625-633

  • DOI: https://doi.org/10.1093/protein/gzh074
  • Primary Citation of Related Structures:  
    1R9X, 1R9Y, 1R9Z, 1RA0, 1RA5, 1RAK

  • 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.


  • Organizational Affiliation

    Department of Pharmaceutical Sciences and the School of Molecular Biosciences, PO Box 646534, Washington State University, Pullman, WA 99164-6534, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Cytosine deaminase430Escherichia coliMutation(s): 2 
Gene Names: CODAB0337
EC: 3.5.4.1
UniProt
Find proteins for P25524 (Escherichia coli (strain K12))
Explore P25524 
Go to UniProtKB:  P25524
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP25524
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.58 Å
  • R-Value Free: 0.184 
  • R-Value Work: 0.164 
  • R-Value Observed: 0.164 
  • Space Group: H 3 2
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 109.037α = 90
b = 109.037β = 90
c = 240.188γ = 120
Software Package:
Software NamePurpose
CNSrefinement
HKL-2000data reduction
SCALEPACKdata scaling
CNSphasing

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2004-10-05
    Type: Initial release
  • Version 1.1: 2008-04-29
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Derived calculations, Version format compliance
  • Version 1.3: 2021-10-27
    Changes: Database references, Derived calculations
  • Version 1.4: 2023-08-23
    Changes: Data collection, Refinement description