Rational design and directed evolution of type 1 deoxyribonucleoside kinases

The importance of type 1 deoxyribonucleoside kinases (dNKs) to the treatment of disease has lead to a great deal of interest in investigating the determinants of substrate specificity of these enzymes, as well as in engineering them to enhance their activity for use in gene therapy applications.; Human thymidine kinase 2 (hTK2) is critical for the nucleotide salvage pathway and phosphorylation of nucleoside analog prodrugs in vivo. hTK2 is strictly pyrimidine-specific, whereas its phylogenetic relative, the Drosophila melanogaster deoxyribonucleoside kinase (D mdNK), shows higher activity and broader specificity towards both pyrimidines and purines. These differences are counterintuitive, as only two of 29 active site residues differ in the two enzymes. I have used site-directed mutagenesis to redesign the hTK2 active site to mimic that of DmdNK, and have characterized the three resulting enzymes (L78F-hTK2, L116M-hTK2 and L78F/L116M-hTK2). These mutations improve the K M for thymidine, increasing the catalytic activity of L78F/L116M-hTK2 4.4-fold, yet leave activity for deoxycytidine and the purine nucleosides unchanged. These results suggest that features other than the non-conserved active site residues are responsible for the observed differences in purine and pyrimidine specificity between these two enzymes.; To further probe the regions of sequence and structure that are important for function and specificity, a directed evolution approach was utilized. Libraries of hybrid enzymes were constructed by non-homologous recombination of hTK2 and DmdNK.; As part of the library construction, an improved system for reading frame selection was developed. The construction and validation of the pInSALect vector, which provides strict reading frame selection without concomitant selection for protein solubility or folding, is described.; From the libraries of hybrid enzymes, I identified chimeras that phosphorylate nucleoside analogs with higher activity than either parental enzyme, and that possess new activity towards the anti-HIV prodrug 2',3'-didehydro-3'-deoxythymidine (d4T). These results demonstrate the potential of non-homologous recombination within the dNK family for creating enzymes with new and improved activities towards nucleoside analogs. In addition, the results presented herein reveal a previously unknown role for the C-terminal regions of these dNKs in determining substrate selectivity.