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.
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Research Article|
May 27 2010
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;
Jean L. Whittingham
1
*Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5YW, U.K.
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Juana Carrero-Lerida;
Juana Carrero-Lerida
1
†Instituto de Parasitología y Biomedicina ‘López-Neyra’, Consejo Superior de Investigaciones Científicas, Parque Tecnológico de Ciencias de la Salud, Avenida del Conocimiento s/n, 18100-Armilla, Granada, Spain
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James A. Brannigan;
James A. Brannigan
*Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5YW, U.K.
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Luis M. Ruiz-Perez;
Luis M. Ruiz-Perez
†Instituto de Parasitología y Biomedicina ‘López-Neyra’, Consejo Superior de Investigaciones Científicas, Parque Tecnológico de Ciencias de la Salud, Avenida del Conocimiento s/n, 18100-Armilla, Granada, Spain
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Ana P. G. Silva;
Ana P. G. Silva
*Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5YW, U.K.
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Mark J. Fogg;
Mark J. Fogg
*Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5YW, U.K.
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Anthony J. Wilkinson;
Anthony J. Wilkinson
*Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5YW, U.K.
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Ian H. Gilbert;
Ian H. Gilbert
‡College of Life Sciences, University of Dundee, Sir James Black Centre, Dundee DD1 5EH, Scotland, U.K.
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Keith S. Wilson;
Keith S. Wilson
2
*Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5YW, U.K.
2Correspondence may be addressed to either of these authors (email dgonzalez@ipb.csic.es or keith@ysbl.york.ac.uk).
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Dolores González-Pacanowska
Dolores González-Pacanowska
2
†Instituto de Parasitología y Biomedicina ‘López-Neyra’, Consejo Superior de Investigaciones Científicas, Parque Tecnológico de Ciencias de la Salud, Avenida del Conocimiento s/n, 18100-Armilla, Granada, Spain
2Correspondence may be addressed to either of these authors (email dgonzalez@ipb.csic.es or keith@ysbl.york.ac.uk).
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Publisher: Portland Press Ltd
Received:
December 10 2009
Revision Received:
March 05 2010
Accepted:
March 30 2010
Accepted Manuscript online:
March 30 2010
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2010 Biochemical Society
2010
Biochem J (2010) 428 (3): 499–509.
Article history
Received:
December 10 2009
Revision Received:
March 05 2010
Accepted:
March 30 2010
Accepted Manuscript online:
March 30 2010
Citation
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 González-Pacanowska; Structural basis for the efficient phosphorylation of AZT-MP (3′-azido-3′-deoxythymidine monophosphate) and dGMP by Plasmodium falciparum type I thymidylate kinase. Biochem J 15 June 2010; 428 (3): 499–509. doi: https://doi.org/10.1042/BJ20091880
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