3G77

Bacterial cytosine deaminase V152A/F316C/D317G mutant

Summary for 3G77

• Entry DOI: 10.2210/pdb3g77/pdb
• Related: 1K6W 1R9Y 1R9Z 1RA0 1RAK 1RB7
• Descriptor: Cytosine deaminase, FE (III) ION (3 entities in total)
• Functional Keywords: cytosine deaminase, protein engineering, cytosine metabolism, hydrolase, iron, metal-binding
• Biological source: Escherichia coli
• Total number of polymer chains: 1
• Total formula weight: 47124.04

Authors

Stoddard, B.,Zhao, L. (deposition date: 2009-02-09, release date: 2009-09-22, Last modification date: 2024-02-21)

Primary citation

Fuchita, M.,Ardiani, A.,Zhao, L.,Serve, K.,Stoddard, B.L.,Black, M.E.
Bacterial cytosine deaminase mutants created by molecular engineering show improved 5-fluorocytosine-mediated cell killing in vitro and in vivo.
Cancer Res., 69:4791-4799, 2009

PubMed Abstract: Cytosine deaminase is used in combination with 5-fluorocytosine as an enzyme-prodrug combination for targeted genetic cancer treatment. This approach is limited by inefficient gene delivery and poor prodrug conversion activities. Previously, we reported individual point mutations within the substrate binding pocket of bacterial cytosine deaminase (bCD) that result in marginal improvements in the ability to sensitize cells to 5-fluorocytosine (5FC). Here, we describe an expanded random mutagenesis and selection experiment that yielded enzyme variants, which provide significant improvement in prodrug sensitization. Three of these mutants were evaluated using enzyme kinetic analyses and then assayed in three cancer cell lines for 5FC sensitization, bystander effects, and formation of 5-fluorouracil metabolites. All variants displayed 18- to 19-fold shifts in substrate preference toward 5FC, a significant reduction in IC(50) values and improved bystander effect compared with wild-type bCD. In a xenograft tumor model, the best enzyme mutant was shown to prevent tumor growth at much lower doses of 5FC than is observed when tumor cells express wild-type bCD. Crystallographic analyses of this construct show the basis for improved activity toward 5FC, and also how two different mutagenesis strategies yield closely related but mutually exclusive mutations that each result in a significant alteration of enzyme specificity.

PubMed: 19487291
DOI: 10.1158/0008-5472.CAN-09-0615

Experimental method

X-RAY DIFFRACTION (1.8 Å)