Non-homologous Recombination of Deoxyribonucleoside Kinases from Human and Drosophila melanogaster Yields Human-like Enzymes with Novel Activities

• Type of Material: Article
• Authors: Monica L. Gerth, Emory University; Stefan Lutz, Emory University
• Language: English
• Date: 2007-07-20
• Publisher: Elsevier
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2007 Elsevier Ltd. All rights reserved.
• License: Creative Commons Attribution-NonCommerical-NoDerivs 3.0 Unported
• Final Published Version (URL): http://dx.doi.org/10.1016/j.jmb.2007.05.021
• Title of Journal or Parent Work: Journal of Molecular Biology
• ISSN: 0022-2836
• Volume: 370
• Issue: 4
• Start Page: 742
• End Page: 751
• Grant/Funding Information: DNA sequencing was performed at the Center for Fundamental and Applied Molecular Evolution at Emory University (NSF-MRI 0320786).; The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: dideoxyinosine (ddI) and Lamivudine (3TC).; This work was supported in part by the National Institutes of Health (GM69958), as well as by a grant to the Emory Center for AIDS Research (AI050409) from the National Institutes of Health and by institutional funding from the Emory University Health Science Center.; M.L.G. is a Teem Family Century ARCS Fellow.
• Keywords: nucleoside analog; human thymidine kinase 2; deoxyribonucleoside kinase; enzyme engineering; substrate specificity; non-homologous recombination
• Research Categories: Chemistry, Biochemistry; Health Sciences, Oncology; Chemistry, General

Abstract

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.