Characterisation of the bifunctional dihydrofolate synthase-folylpolyglutamate synthase from Plasmodium falciparum; a potential novel target for antimalarial antifolate inhibition

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 0.50 μM and 1.25 μM, respectively. DHFS activity measured under lower co-substrate concentrations, where data fitted the Michaelis-Menten equation, yielded apparent Km values of 0.88 μM for DHP, 22.8 μM for ATP and 5.97 μM for glutamate. Of the substrates tested in FPGS assays, only tetrahydrofolate (THF) was efficiently converted to polyglutamylated forms, exhibiting standard kinetics with an apparent Km of 0.96 μM; 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 Ki value of an aryl phosphinate analogue against DHFS was 0.14 μM and for an alkyl phosphinate against FPGS 0.091 μM, 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.

Graphical abstractThe activities of the bifunctional folate pathway enzyme dihydrofolate synthase-folylpolyglutamate synthase from Plasmodium falciparum are characterised with respect to their kinetics, substrate specificities and responses to folate analogue inhibitors.Download high-res image (32KB)Download full-size image