title: Characterisation of the bifunctional dihydrofolate synthase-folylpolyglutamate synthase from Plasmodium falciparum; a potential novel target for antimalarial antifolate inhibition.
abstract: Unusually for a eukaryote, the malaria parasite Plasmodium falciparum expresses dihydrofolate synthase (DHFS) and folylpolyglutamate synthase (FPGS) as a single bifunctional protein. The two activities contribute to the essential pathway of folate biosynthesis and modification. The DHFS activity of recombinant PfDHFS-FPGS exhibited non-standard kinetics at high co-substrate (glutamate and ATP) concentrations, being partially inhibited by increasing concentrations of its principal substrate, dihydropteroate (DHP). Binding of DHP to the catalytic and inhibitory sites exhibited dissociation constants of 0.50microM and 1.25microM, respectively. DHFS activity measured under lower co-substrate concentrations, where data fitted the Michaelis-Menten equation, yielded apparent K(m) values of 0.88microM for DHP, 22.8microM for ATP and 5.97microM for glutamate. Of the substrates tested in FPGS assays, only tetrahydrofolate (THF) was efficiently converted to polyglutamylated forms, exhibiting standard kinetics with an apparent K(m) of 0.96microM; dihydrofolate, folate and the folate analogue methotrexate (MTX) were negligibly processed, emphasising the importance of the oxidation state of the pterin moiety. Moreover, MTX inhibited neither DHFS nor FPGS, even at high concentrations. Conversely, two phosphinate analogues of 7,8-dihydrofolate that mimic tetrahedral intermediates formed during DHFS- and FPGS-catalysed glutamylation were powerfully inhibitory. The K(i) value of an aryl phosphinate analogue against DHFS was 0.14microM and for an alkyl phosphinate against FPGS 0.091microM, with each inhibitor showing a high degree of specificity. This, combined with the absence of DHFS activity in humans, suggests PfDHFS-FPGS might represent a potential new drug target in the previously validated folate pathway of P. falciparum.
authors.affiliation: Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.
authors.forename: Ping
authors.initials: P
authors.lastname: Wang
authors.forename: Qi
authors.initials: Q
authors.lastname: Wang
authors.forename: Yonghong
authors.initials: Y
authors.lastname: Yang
authors.forename: James K
authors.initials: JK
authors.lastname: Coward
authors.forename: Alexis
authors.initials: A
authors.lastname: Nzila
authors.forename: Paul F G
authors.initials: PF
authors.lastname: Sims
authors.forename: John E
authors.initials: JE
authors.lastname: Hyde
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D004791:Enzyme Inhibitors; D005492:Folic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D007700:Kinetics; D008727:Methotrexate / Q000378:metabolism; D010453:Peptide Synthases / Q000378:metabolism*; D010963:Plasmodium falciparum / Q000201:enzymology*; D015800:Protozoan Proteins / Q000378:metabolism*; D011622:Pterins / Q000378:metabolism; D013379:Substrate Specificity; D013763:Tetrahydrofolates / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D015800:Protozoan Proteins; D011622:Pterins; D013763:Tetrahydrofolates; C000978:dihydropteroate; D018698:Glutamic Acid; C010920:dihydrofolate; C030371:5,6,7,8-tetrahydrofolic acid; D000255:Adenosine Triphosphate; D005492:Folic Acid; D010453:Peptide Synthases; C020041:dihydrofolate synthetase; C023103:folylpolyglutamate synthetase; D008727:Methotrexate