Active site mutants of Drosophila melanogaster multisubstrate deoxyribonucleoside kinase.
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.
Solaroli
Nicola
N
Division of Clinical Virology F68, Karolinska Institute, Huddinge University Hospital, Stockholm, Sweden and Dipartimento di Scienze Farmaceutiche, Università di Ferrara, Italy. nicola.solaroli@labmed.ki.se
Bjerke
Mia
M
Amiri
Marjan H
MH
Johansson
Magnus
M
Karlsson
Anna
A
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009705:Nucleosides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment
D019476:Insect Proteins; D009705:Nucleosides; D017853:Phosphotransferases (Alcohol Group Acceptor); C020018:deoxyribonucleoside kinases