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Active site mutants of Drosophila melanogaster multisubstrate deoxyribonucleoside kinase
Nicola Solaroli1, Mia Bjerke, Marjan H Amiri
1Division 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
Abstract:
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.
Insights
Researchers modified Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) to improve purine phosphorylation for suicide gene therapy. A Q81N mutation enhanced deoxyguanosine phosphorylation but reduced deoxythymidine phosphorylation, indicating key roles for specific residues.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzyme Engineering
Background:
- The multisubstrate deoxyribonucleoside kinase (Dm-dNK) from Drosophila melanogaster shares sequence similarity with human deoxyribonucleoside kinases and viral thymidine kinases.
- Dm-dNK exhibits broad substrate specificity, phosphorylating both purine and pyrimidine deoxyribonucleosides and nucleoside analogs, with a preference for pyrimidines.
Purpose of the Study:
- To engineer Dm-dNK for enhanced purine nucleoside phosphorylation efficiency.
- To develop an improved enzyme for potential applications in suicide gene therapy.
Main Methods:
- Site-directed mutagenesis was employed to alter residues involved in substrate recognition, guided by structural data.
- Specific mutations, including Q81N and substitutions at N28, I29, and F114, were introduced and characterized.
Main Results:
- The Q81N mutation resulted in a relative increase in deoxyguanosine phosphorylation compared to the wild-type enzyme.
- This mutation led to a significant decrease (10-fold) in deoxythymidine phosphorylation efficiency.
- Mutations at N28, I29, and F114 also decreased thymidine phosphorylation, underscoring their importance in substrate binding and catalysis.
Conclusions:
- Specific amino acid residues, such as Q81, N28, I29, and F114, play critical roles in the substrate specificity and catalytic activity of Dm-dNK.
- Targeted mutagenesis can alter enzyme activity, but achieving desired improvements requires careful consideration of residue function.