Structural and kinetic characterization of human deoxycytidine kinase variants able to phosphorylate 5-substituted deoxycytidine and thymidine analogues .
The physiological role of human deoxycytidine kinase (dCK) is to phosphorylate deoxynucleosides required for DNA synthesis, with the exception of thymidine. Previous structural analysis of dCK implicated steric factors, specifically the thymine methyl group at the 5-position, that prevent thymidine phosphorylation by dCK. This hypothesis is supported by the observation that mutations that enlarge the active site cavity in proximity to the nucleoside 5-position endow dCK with the ability to phosphorylate thymidine. However, in conflict with this hypothesis was our discovery that the cytidine analogue 5-methyldeoxycytidine (5-Me-dC), an isostere of thymidine, can indeed be phosphorylated by wild-type (WT) dCK. To reconcile this seemingly contradicting observation, and to better understand the determinants preventing thymidine phosphorylation by WT dCK, we solved the crystal structure of dCK in complex with 5-Me-dC. The structure reveals the active site adjustments required to accommodate the methyl group at the 5-position. Combination of kinetic, mutagenesis, and structural data suggested that it is in fact residue Asp133 of dCK that is most responsible for discriminating against the thymine base. dCK variants in which Asp133 is replaced by an alanine and Arg104 by select hydrophobic residues attain significantly improved activity with 5-substituted deoxycytidine and thymidine analogues. Importantly, the ability of the designer enzymes to activate 5-substitued pyrimidines makes it possible to utilize such nucleoside analogues in suicide gene therapy or protein therapy applications that target cancer cells.
Hazra
Saugata
S
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, 900 South Ashland Avenue, Chicago, Illinois 60607, USA.
Ort
Stephan
S
Konrad
Manfred
M
Lavie
Arnon
A
D019943:Amino Acid Substitution; D018360:Crystallography, X-Ray; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000378:metabolism*; D003842:Deoxycytidine Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D010766:Phosphorylation; D013379:Substrate Specificity; D013936:Thymidine / Q000031:analogs & derivatives / Q000378:metabolism*
D003841:Deoxycytidine; C016569:5-methyldeoxycytidine; D003842:Deoxycytidine Kinase; D013936:Thymidine