國家衛生研究院 NHRI:Item 3990099045/2857
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    题名: Identification and characterization of the conserved nucleoside-binding sites in the Epstein-Barr virus thymidine kinase
    作者: Wu, CC;Chen, MC;Chang, YR;Hsu, TY;Chen, JY
    贡献者: National Institute of Cancer Research
    摘要: Thymidine kinase TK, encoded by EBV (Epstein-Baff 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 (Asp(392) --> Glu) and R393H retain activity, indicating that a negative charge is important for Asp(392) and a positive charge is required for Arg(392). 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 Asp(392) plays multiple roles in this region. His(394) 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 Phe(402) is substituted with serine residue, the original preferred pyrimidine substrates, such as Y-azido-Y-deoxythymidine, iododeoxyuridine and beta-L-5-iododioxolane uracil (L-form substrate), have decreased competitiveness with thymidine, suggesting that Phe(402). plays a crucial role in substrate specificity and that the aromatic ring is important for function.
    关键词: Biochemistry & Molecular Biology
    日期: 2004-05-01
    關聯: Biochemical Journal. 2004 May;379(Pt. 3):795-803.
    Link to: http://dx.doi.org/10.1042/BJ20031832
    JIF/Ranking 2023: http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=NHRI&SrcApp=NHRI_IR&KeyISSN=0264-6021&DestApp=IC2JCR
    Cited Times(WOS): https://www.webofscience.com/wos/woscc/full-record/WOS:000221535000032
    Cited Times(Scopus): http://www.scopus.com/inward/record.url?partnerID=HzOxMe3b&scp=2542565697
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