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

TY - JOUR

T1 - Structural basis for the efficient phosphorylation of AZT-MP (3 '-azido-3 '-deoxythymidine monophosphate) and dGMP by Plasmodium falciparum type I thymidylate kinase

AU - Whittingham, Jean L.

AU - Carrero-Lerida, Juana

AU - Brannigan, James A.

AU - Ruiz-Perez, Luis M.

AU - Silva, Ana P. G.

AU - Fogg, Mark J.

AU - Wilkinson, Anthony J.

AU - Gilbert, Ian H.

AU - Wilson, Keith S.

AU - Gonzalez-Pacanowska, Dolores

PY - 2010/6/15

Y1 - 2010/6/15

N2 - 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.

AB - 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.

KW - drug target

KW - ligand complex

KW - malaria

KW - Plasmodium falciparum

KW - thymidylate kinase (TMPK)

KW - CRYSTAL-STRUCTURE

KW - SUBSTRATE-SPECIFICITY

KW - MOLECULAR-GRAPHICS

KW - UMP KINASE

KW - TMP KINASE

KW - ACTIVATION

KW - ANALOGS

KW - BINDING

KW - INHIBITION

KW - EXPRESSION

U2 - 10.1042/BJ20091880

DO - 10.1042/BJ20091880

M3 - Article

C2 - 20353400

SN - 0264-6021

VL - 428

SP - 499

EP - 509

JO - Biochemical Journal

JF - Biochemical Journal

ER -