Structural basis for the changed substrate specificity of Drosophila melanogaster deoxyribonucleoside kinase mutant N64D.
The Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) double mutant N45D/N64D was identified during a previous directed evolution study. This mutant enzyme had a decreased activity towards the natural substrates and decreased feedback inhibition with dTTP, whereas the activity with 3'-modified nucleoside analogs like 3'-azidothymidine (AZT) was nearly unchanged. Here, we identify the mutation N64D as being responsible for these changes. Furthermore, we crystallized the mutant enzyme in the presence of one of its substrates, thymidine, and the feedback inhibitor, dTTP. The introduction of the charged Asp residue appears to destabilize the LID region (residues 167-176) of the enzyme by electrostatic repulsion and no hydrogen bond to the 3'-OH is made in the substrate complex by Glu172 of the LID region. This provides a binding space for more bulky 3'-substituents like the azido group in AZT but influences negatively the interactions between Dm-dNK, substrates and feedback inhibitors based on deoxyribose. The detailed picture of the structure-function relationship provides an improved background for future development of novel mutant suicide genes for Dm-dNK-mediated gene therapy.
Welin
Martin
M
Department of Molecular Biology, Swedish University of Agricultural Sciences, Biomedical Center, Uppsala, Sweden.
Skovgaard
Tine
T
Knecht
Wolfgang
W
Zhu
Chunying
C
Berenstein
Dvora
D
Munch-Petersen
Birgitte
B
Piskur
Jure
J
Eklund
Hans
H
D000818:Animals; D001216:Asparagine / Q000235:genetics* / Q000378:metabolism; D001224:Aspartic Acid / Q000235:genetics* / Q000378:metabolism; D018360:Crystallography, X-Ray; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics*; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D013942:Thymine Nucleotides / Q000494:pharmacology
D013942:Thymine Nucleotides; D001224:Aspartic Acid; D001216:Asparagine; D017853:Phosphotransferases (Alcohol Group Acceptor); C020018:deoxyribonucleoside kinases; C024157:thymidine 5'-triphosphate