Modifying human thymidylate kinase to potentiate azidothymidine activation

Based on the knowledge of the crystal structures of yeast and Escherichia coli thymidylate kinases (TmpKs) and the observation that TmpK from E. coli can phosphorylate azidothymidine monophosphate (AZT-MP) much more efficiently than either the yeast or the highly homologous human enzyme, we have engineered yeast and human TmpKs to obtain enzymes that have dramatically improved AZT-MP phosphorylation properties. These modified enzymes have properties that make them attractive candidates for gene therapeutic approaches to potentiating the action of AZT as an inhibitor of human immunodeficiency virus (HIV) replication. In particular, insertion of the lid domain of the bacterial TmpK into the human enzyme results in a pronounced change of the acceptance of AZT-MP such that it is now phosphorylated even faster than TMP. One of the main reasons for the less than optimal properties of nucleoside prodrugs in the treatment of HIV1 infection is their poor phosphorylation to the active triphosphate form by cellular enzymes. In the case of 3-azido-3-deoxythymidine (AZT), the bottleneck in its activation appears to be addition of the second phosphoryl group to AZT-monophosphate (AZT-MP) by thymidylate kinase (TmpK) (1, 2). Earlier work suggested that a specific and unique feature of the highly homologous yeast and human TmpKs (Fig. 1) is responsible for the low efficiency of these enzymes in phosphorylating AZT-MP. This is the fact that an arginine at position X3 of the P-loop (the GX1X2X3X4GKS(T) motif involved in fixing the α- and β-phosphates of ATP (3)) appears to play an important catalytic role but is mispositioned because of steric hindrance between the azido group of AZT-MP and the preceding carboxylic acid side chain of Asp-14 (position X2) (4, 5). A catalytic role was assigned to Arg-15 of yeast TmpK based on the observations that in the structure with the bisubstrate inhibitor P1-(5-adenosyl)-P5-(5-thymidyl)pentaphosphate (TP5A) this residue interacts with the γ-phosphate of the ATP moiety and that its mutation to glycine results in a 200-fold decrease of phosphorylation activity with the natural substrate, dTMP (Table I). Interestingly, TmpK from E. coli has no arginine in its P-loop motif but rather several arginine residues in its lid region, a flexible stretch of amino acids that in adenylate kinase becomes ordered upon ATP binding (6). One or more of these arginines in E. coli TmpK were predicted to assume the catalytic role assigned to the P-loop arginine of the yeast enzyme. Therefore, we anticipated that the bacterial TmpK would be less affected by the presence of the azido group in AZT-MP, and this prediction proved to be correct (7). The crystal structure of the E. coli enzyme suggested that the role of Arg-15 in the yeast enzyme is taken over by Arg-153 from the lid region (8). Using this knowledge we have engineered yeast and human TmpKs to obtain enzymes that have dramatically improved AZT-MP phosphorylation properties