tailieunhanh - Báo cáo khoa học: Active site mutants of Drosophila melanogaster multisubstrate deoxyribonucleoside kinase

The multisubstrate deoxyribonucleoside kinase ofDroso-phila melanogaster (Dm-dNK) is sequence-related to three human deoxyribonucleoside kinases and to herpes simplex virus type-1 thymidine kinase. Dm-dNK phos-phorylates both purine and pyrimidine deoxyribonucleo-sides 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 sub-stratestocreateanimprovedenzymetobeusedinsuicide gene therapy. . | Eur. J. Biochem. 270 2879-2884 2003 FEBS 2003 doi Active site mutants of Drosophila melanogaster multisubstrate deoxyribonucleoside kinase Nicola Solaroli1 2 Mia Bjerke1 Marjan H. Amiri1 Magnus Johansson1 and Anna Karlsson1 Division of Clinical Virology F68 Karolinska Institute Huddinge University Hospital Stockholm Sweden 2Dipartimento di Scienze Farmaceutiche Universita di Ferrara Ferrara Italy 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 wildtype enzyme supportingtheir importance for substrate bindingand or catalysis as proposed by the recently solved structure of Dm-dNK. Keywords gene therapy nucleoside analog suicide gene. The deoxyribonucleoside kinase of the fruit fly Drosophila melanogaster Dm-dNK is a multisubstrate enzyme that phosphorylates pyrimidine and purine deoxyribonucleosides as well as several anticancer and antiviral nucleoside analogues 1-3 . Dm-dNK is sequence-related to the human deoxycytidine kinase dCK deoxyguanosine kinase