Thymidine kinase (TK), encoded by EBV (Epstein–Barr virus), is an attractive target for antiviral therapy and provides a novel approach to the treatment of EBV-associated malignancies. Despite the extensive use of nucleoside analogues for the treatment of viral infections and cancer, the structure–function relationship of EBV TK has been addressed rarely. In the absence of any structural information, we sought to identify and elucidate the functional roles of amino acids in the nucleoside-binding site using site-directed mutagenesis. Through alignment with other human herpesviral TK protein sequences, we predicted that certain conserved regions comprise the nucleoside-binding site of EBV TK and, through site-directed mutagenesis, showed significant changes in activity and binding affinity for thymidine of site 3 (-DRH-) and 4 (-VFP-) mutants. For site 3, only mutants D392E (Asp392→Glu) and R393H retain activity, indicating that a negative charge is important for Asp392 and a positive charge is required for Arg393. The increased binding affinities of these two mutants for 3´-deoxy-2´,3´-didehydrothymidine suggest that the two residues are also important for substrate selection. Interestingly, the changed metal-ion usage pattern of D392E reveals that Asp392 plays multiple roles in this region. His394 cannot be compensated by other amino acids, also indicating a crucial role. In site 4, the F402Y mutant retains full activity; however, F402S retains only 60% relative activity. Strikingly, when Phe402 is substituted with serine residue, the original preferred pyrimidine substrates, such as 3´-azido-3´-deoxythymidine, iododeoxyuridine and β-l-5-iododioxolane uracil (l-form substrate), have decreased competitiveness with thymidine, suggesting that Phe402 plays a crucial role in substrate specificity and that the aromatic ring is important for function.
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Research Article|
May 01 2004
Identification and characterization of the conserved nucleoside-binding sites in the Epstein-Barr virus thymidine kinase
Chung-Chun WU;
Chung-Chun WU
*Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Jen-Ai Road, Taipei 100, Taiwan, Republic of China
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Min-Che CHEN;
Min-Che CHEN
*Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Jen-Ai Road, Taipei 100, Taiwan, Republic of China
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Ya-Ru CHANG;
Ya-Ru CHANG
*Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Jen-Ai Road, Taipei 100, Taiwan, Republic of China
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Tsuey-Ying HSU;
Tsuey-Ying HSU
1
*Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Jen-Ai Road, Taipei 100, Taiwan, Republic of China
1Correspondence may be addressed to either of the authors (e-mail tyhsu@ha.mc.ntu.edu.tw or cjy@nhri.org.tw).
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Jen-Yang CHEN
Jen-Yang CHEN
1
*Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Jen-Ai Road, Taipei 100, Taiwan, Republic of China
†Extramural Research Affairs Department, National Health Research Institutes, Min-Chuan East Road, Taipei 114, Taiwan, Republic of China
1Correspondence may be addressed to either of the authors (e-mail tyhsu@ha.mc.ntu.edu.tw or cjy@nhri.org.tw).
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Publisher: Portland Press Ltd
Received:
November 28 2003
Accepted:
January 05 2004
Accepted Manuscript online:
January 05 2004
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2004
2004
Biochem J (2004) 379 (3): 795–803.
Article history
Received:
November 28 2003
Accepted:
January 05 2004
Accepted Manuscript online:
January 05 2004
Citation
Chung-Chun WU, Min-Che CHEN, Ya-Ru CHANG, Tsuey-Ying HSU, Jen-Yang CHEN; Identification and characterization of the conserved nucleoside-binding sites in the Epstein-Barr virus thymidine kinase. Biochem J 1 May 2004; 379 (3): 795–803. doi: https://doi.org/10.1042/bj20031832
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