Structural basis for the efficient phosphorylation of AZT-MP (3 '-azido-3 '-deoxythymidine monophosphate) and dGMP by Plasmodium falciparum type I thymidylate kinase

Jean L. Whittingham, Juana Carrero-Lerida, James A. Brannigan, Luis M. Ruiz-Perez, Ana P. G. Silva, Mark J. Fogg, Anthony J. Wilkinson, Ian H. Gilbert, Keith S. Wilson, Dolores Gonzalez-Pacanowska

    Research output: Contribution to journalArticle

    27 Citations (Scopus)

    Abstract

    Plasmodium falciparum is the causative agent of malaria, a disease where new drug targets are required due to increasing resistance to current anti-malarials. TMPK (thymidylate kinase) is a good candidate as 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 AZT (3'-azido3'-deoxythymidine)-MP (monophosphate) inefficiently compared with type II TMPKs (e.g. Escherichia coli TMPK). In the present paper we report that eukaryotic PfTMPK (P falciparum TMPK) presents sequence features of a type I enzyme yet the kinetic parameters for AZT-MP 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 AZT-MP. 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 results that confirm the ability of the enzyme to uniquely accept dGMP as a substrate and shed light on the basis for its wider substrate specificity. Information resulting from two ternary complexes (dTMP ADP and AZT-MP ADP) and a binary complex with the transition state analogue AP(5)dT [P-1-(5'-adenosyl)-P-5-(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.

    Original languageEnglish
    Pages (from-to)499-509
    Number of pages11
    JournalBiochemical Journal
    Volume428
    DOIs
    Publication statusPublished - 15 Jun 2010

    Keywords

    • drug target
    • ligand complex
    • malaria
    • Plasmodium falciparum
    • thymidylate kinase (TMPK)
    • CRYSTAL-STRUCTURE
    • SUBSTRATE-SPECIFICITY
    • MOLECULAR-GRAPHICS
    • UMP KINASE
    • TMP KINASE
    • ACTIVATION
    • ANALOGS
    • BINDING
    • INHIBITION
    • EXPRESSION

    Cite this

    Whittingham, J. L., Carrero-Lerida, J., Brannigan, J. A., Ruiz-Perez, L. M., Silva, A. P. G., Fogg, M. J., Wilkinson, A. J., Gilbert, I. H., Wilson, K. S., & Gonzalez-Pacanowska, D. (2010). Structural basis for the efficient phosphorylation of AZT-MP (3 '-azido-3 '-deoxythymidine monophosphate) and dGMP by Plasmodium falciparum type I thymidylate kinase. Biochemical Journal, 428, 499-509. https://doi.org/10.1042/BJ20091880