Structural basis for the efficient phosphorylation of AZT-MP (3'-azido-3'-deoxythymidine monophosphate) and dGMP by Plasmodium falciparum type I thymidylate kinase.
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.
Whittingham
Jean L
JL
Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO105YW, UK.
Carrero-Lerida
Juana
J
Brannigan
James A
JA
Ruiz-Perez
Luis M
LM
Silva
Ana P G
AP
Fogg
Mark J
MJ
Wilkinson
Anthony J
AJ
Gilbert
Ian H
IH
Wilson
Keith S
KS
González-Pacanowska
Dolores
D
D003848:Deoxyguanine Nucleotides / Q000737:chemistry / Q000378:metabolism*; D054306:Dideoxynucleotides / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D009703:Nucleoside-Phosphate Kinase / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000378:metabolism; D013379:Substrate Specificity; D013942:Thymine Nucleotides / Q000737:chemistry* / Q000378:metabolism*; D015215:Zidovudine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
D003848:Deoxyguanine Nucleotides; D054306:Dideoxynucleotides; D013942:Thymine Nucleotides; C057287:3'-azido-3'-deoxythymidine 5'phosphate; D015215:Zidovudine; C007257:2'-deoxyguanosine 5'-phosphate; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase