Structural basis for the efficient phosphorylation of AZTMP and dGMP by Plasmodium falciparum type I thymidylate kinase.
Résumé
Plasmodium falciparum is the causative agent of malaria, a disease where new drug targets are required due to increasing resistance to current anti-malarials. Thymidylate kinase (TMPK) is a good candidate since it is essential for the synthesis of dTTP, a critical precursor of DNA and has been much studied due to its role in prodrug activation and as a drug target. Type I TMPKs such as the human enzyme, phosphorylate the substrate 3'-azido-3'-deoxythymidine monophosphate (AZTMP) inefficiently compared to type II TMPKs (e.g. E. coli TMPK). Here we report that eukaryotic P. falciparum TMPK presents sequence features of a type I enzyme yet kinetic parameters for AZTMP phosphorylation are similar to those of the highly efficient E. coli enzyme. Structural information shows that this is explained by a different juxtaposition of the P-loop and the azide of AZTMP. Subsequent formation of the transition state requires no further movement of the PfTMPK P-loop, with no steric conflicts for the azide moiety, allowing efficient phosphate transfer. Likewise, we present data that confirms the ability of the enzyme to accept uniquely dGMP as a substrate and sheds light on the basis for its wider substrate specificity. Information resulting from two ternary complexes (dTMP-ADP and AZTMP-ADP) and a binary complex with the transition state analogue P1-(5'-adenosyl)-P5-(5'-thymidyl) pentaphosphate all reveal significant differences with the human enzyme notably in the lid region and in the P-loop which may be exploited in the rational design of Plasmodium-specific TMPK inhibitors with therapeutic potential.
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