Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 370, Issue 4, Pages 742-751Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2007.05.021
Keywords
enzyme engineering; kinase; nucleoside analog
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Funding
- NIAID NIH HHS [P30 AI050409, AI050409] Funding Source: Medline
- NIGMS NIH HHS [GM69958, R01 GM069958, R01 GM069958-03] Funding Source: Medline
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In antiviral and cancer therapy, deoxyribonucleoside kinases (dNKs) are often the rate-limiting step in activating nucleoside analog (NA) prodrugs into their cytotoxic, phosphorylated forms. We have constructed libraries of hybrid enzymes by non-homologous recombination of the pyrimidine-specific human thymidine kinase 2 and the broad-specificity dNK from Drosophila melanogaster; their low sequence identity has precluded engineering by conventional, homology-dependent shuffling techniques. From these libraries, we identified chimeras that phosphorylate nucleoside analogs with higher activity than either parental enzyme, and that possess new activity towards the anti-HIV prodrug 2',3'-didehydro-3'-deoxythymidine (d4T). These results demonstrate the potential of non-homologous recombination within the dNK family for creating enzymes with new and improved activities towards nucleoside analogs. In addition, our results exposed a previously unknown role for the C-terminal regions of these dNKs in determining substrate selectivity. (c) 2007 Elsevier Ltd. All rights reserved.
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