Solaroli Nicola, Bjerke Mia, Amiri Marjan H, Johansson Magnus, Karlsson Anna
Division of Clinical Virology F68, Karolinska Institute, Huddinge University Hospital, Stockholm, Sweden and Dipartimento di Scienze Farmaceutiche, Università di Ferrara, Italy.
Eur J Biochem. 2003 Jul;270(13):2879-84. doi: 10.1046/j.1432-1033.2003.03666.x.
The multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK) is sequence-related to three human deoxyribonucleoside kinases and to herpes simplex virus type-1 thymidine kinase. Dm-dNK phosphorylates both purine and pyrimidine deoxyribonucleosides and nucleoside analogues although it has a preference for pyrimidine nucleosides. We performed site-directed mutagenesis on residues that, based on structural data, are involved in substrate recognition. The aim was to increase the phosphorylation efficiency of purine nucleoside substrates to create an improved enzyme to be used in suicide gene therapy. A Q81N mutation showed a relative increase in deoxyguanosine phosphorylation compared with the wild-type enzyme although the efficiency of deoxythymidine phosphorylation was 10-fold lower for the mutant. In addition to residue Q81 the function of amino acids N28, I29 and F114 was investigated by different substitutions. All of the mutated enzymes showed decreased efficiency of thymidine phosphorylation in comparison with the wild-type enzyme supporting their importance for substrate binding and/or catalysis as proposed by the recently solved structure of Dm-dNK.
果蝇（Drosophila melanogaster）的多底物脱氧核糖核苷激酶（Dm-dNK）与三种人类脱氧核糖核苷激酶以及单纯疱疹病毒1型胸苷激酶在序列上相关。Dm-dNK可磷酸化嘌呤和嘧啶脱氧核糖核苷以及核苷类似物，不过它对嘧啶核苷更具偏好性。我们基于结构数据，对参与底物识别的残基进行了定点诱变。目的是提高嘌呤核苷底物的磷酸化效率，从而创造一种改良酶用于自杀基因治疗。与野生型酶相比，Q81N突变体的脱氧鸟苷磷酸化相对增加，不过该突变体的脱氧胸苷磷酸化效率比野生型低10倍。除了Q81残基外，还通过不同的取代对氨基酸N28、I29和F114的功能进行了研究。与野生型酶相比，所有突变酶的胸苷磷酸化效率均降低，这支持了最近解析的Dm-dNK结构所提出的这些残基对于底物结合和/或催化的重要性。
