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

DOI: 10.1042/BJ20091880 DOI Help

Authors: Jean L. Whittingham (University of York) , Juanna Carrero‑lerida (Instituto de Parasitología y Biomedicina ‘López-Neyra’) , James A. Brannigan (University of York) , Luis M. Ruiz-perez (Instituto de Parasitología y Biomedicina ‘López-Neyra’) , Ana P. G. Silva (University of Manchester) , Mark J. Fogg (University of York) , Anthony J. Wilkinson (University of York) , Ian H. Gilbert (University of Dundee) , Keith S. Wilson (University of York) , Dolores González-pacanowska (Instituto de Parasitología y Biomedicina ‘López-Neyra’)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Biochemical Journal , VOL 428 (3) , PAGES 499–509

State: Published (Approved)
Published: May 2010

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′-azido-3′-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 AP5dT [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.

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: I24-Microfocus Macromolecular Crystallography