期刊
JOURNAL OF MOLECULAR BIOLOGY
卷 377, 期 3, 页码 854-869出版社
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2008.01.002
关键词
thermostability; prodrug gene therapy; cytosine deaminase; random mutagenesis; structure
资金
- NCI NIH HHS [R01 CA085939-08, R01 CA085939, R01 CA097328-03, CA97328, R01 CA085939-01, R01 CA097328-05A1, R01 CA097328-04, R01 CA085939-09, R01 CA097328, R01 CA097328-01A1, CA85939, R01 CA085939-07, R01 CA085939-06, R01 CA085939-03, R01 CA085939-02, R01 CA097328-02, R01 CA085939-05, R01 CA085939-04] Funding Source: Medline
- NIGMS NIH HHS [T32 GM008268, T32 GM008268-18, T32-GM08268, T32 GM008268-19, T32 GM008268-17] Funding Source: Medline
Prodrug gene therapy (PGT) is a treatment strategy in which tumor cells are transfected with a 'suicide' gene that encodes a metabolic enzyme capable of converting a nontoxic prodrug into a potent cytotoxin. One of the most promising PGT enzymes is cytosine deaminase (CD), a microbial salvage enzyme that converts cytosine to uracil. CD also converts 5-fluorocytosine (5FC) to 5-fluorouracil, an inhibitor of DNA synthesis and RNA function. Over 150 studies of CD-mediated PGT applications have been reported since 2000, all using wild-type enzymes. However, various forms of CD are limited by inefficient turnover of 5FC and/or limited thermostability. In a previous study, we stabilized and extended the half-life of yeast CD (yCD) by repacking of its hydrophobic core at several positions distant from the active site. Here we report that random mutagenesis of residues selected based on alignment with similar enzymes, followed by selection for enhanced sensitization to 5FC, also produces an enzyme variant (yCD-D92E) with elevated T-m values and increased activity half-life. The new mutation is located at the enzyme's dimer interface, indicating that independent mutational pathways can lead to an increase in stability, as well as a more subtle effect on enzyme kinetics. Each independently derived set of mutations significantly improves the enzyme's performance in PGT assays both in cell culture and in animal models. (C) 2008 Elsevier Ltd. All rights reserved.
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