Abstract
Folate metabolism in malaria parasites is a long-standing, clinical target for chemotherapy and prophylaxis. However, despite determination of the complete genome sequence of the lethal species Plasmodium falciparum, the pathway of de novo folate biosynthesis remains incomplete, as no candidate gene for dihydroneopterin aldolase (DHNA) could be identified. This enzyme catalyses the third step in the well-characterized pathway of plants, bacteria, and those eukaryotic microorganisms capable of synthesizing their own folate. Utilizing bioinformatics searches based on both primary and higher protein structures, together with biochemical assays, we demonstrate that P. falciparum cell extracts lack detectable DHNA activity, but that the parasite possesses an unusual orthologue of 6-pyruvoyltetrahydropterin synthase (PTPS), which simultaneously gives rise to two products in comparable amounts, the predominant of which is 6-hydroxymethyl-7,8-dihydropterin, the substrate for the fourth step in folate biosynthesis (catalysed by 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase; PPPK). This can provide a bypass for the missing DHNA activity and thus a means of completing the biosynthetic pathway from GTP to dihydrofolate. Supported by site-directed mutagenesis experiments, we ascribe the novel catalytic activity of the malarial PTPS to a Cys to Glu change at its active site relative to all previously characterized PTPS molecules, including that of the human host.
Highlights
Folate cofactors are essential molecules for all living organisms, required for the transfer of one-carbon units in a number of metabolic steps, including the key methylation of dUMP to give dTMP, an essential nucleotide for DNA synthesis
Utilizing bioinformatics searches based on both primary and higher protein structures, together with biochemical assays, we demonstrate that P. falciparum cell extracts lack detectable dihydroneopterin aldolase (DHNA) activity, but that the parasite possesses an unusual orthologue of 6-pyruvoyltetrahydropterin synthase (PTPS), which simultaneously gives rise to two products in comparable amounts, the predominant of which is 6-hydroxymethyl-7,8-dihydropterin, the substrate for the fourth step in folate biosynthesis
This can provide a bypass for the missing DHNA activity and a means of completing the biosynthetic pathway from GTP to dihydrofolate
Summary
Folate cofactors are essential molecules for all living organisms, required for the transfer of one-carbon units in a number of metabolic steps, including the key methylation of dUMP to give dTMP, an essential nucleotide for DNA synthesis. Most microorganisms can synthesize the required folates from the simple precursors GTP, p-aminobenzoate (pABA) and glutamate. The purine ring system of GTP is rearranged by GTP cyclohydrolase I (GTPCH-I; EC 3.5.4.16) to that of a pterin (7,8dihydroneopterin triphosphate; DHNTP), the three-carbon side-chain of which is subsequently cleaved to leave one carbon atom, after which pABA and glutamate are linked to the resulting pterin (6-hydroxymethyl-7,8 dihydropterin; 6HMDP) in successive steps involving 6-hydroxymethyl7,8-dihydropterin pyrophosphokinase (PPPK or HPPK; EC 2.7.6.3), dihydropteroate synthase (DHPS; EC 2.5.1.15) and dihydrofolate synthase (DHFS; EC 6.3.2.12) [Fig. 1, scheme (a)]. A long-standing mystery concerning this pathway in P. falciparum is the apparent lack of a gene encoding the enzyme required for the third step in this pathway, dihydroneopterin aldolase (DHNA; EC 4.1.2.25), 610 S.
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