Plasmodium serine hydroxymethyltransferase as a potential anti-malarial target: inhibition studies using improved methods for enzyme production and assay

Abstract

BackgroundThere is an urgent need for the discovery of new anti-malarial drugs. Thus, it is essential to explore different potential new targets that are unique to the parasite or that are required for its viability in order to develop new interventions for treating the disease. Plasmodium serine hydroxymethyltransferase (SHMT), an enzyme in the dTMP synthesis cycle, is a potential target for such new drugs, but convenient methods for producing and assaying the enzyme are still lacking, hampering the ability to screen inhibitors.MethodsProduction of recombinant Plasmodium falciparum SHMT (PfSHMT) and Plasmodium vivax SHMT (PvSHMT), using auto-induction media, were compared to those using the conventional Luria Bertani medium with isopropyl thio-β-D-galactoside (LB-IPTG) induction media. Plasmodium SHMT activity, kinetic parameters, and response to inhibitors were measured spectrophotometrically by coupling the reaction to that of 5,10-methylenetetrahydrofolate dehydrogenase (MTHFD). The identity of the intermediate formed upon inactivation of Plasmodium SHMTs by thiosemicarbazide was investigated by spectrophotometry, high performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC-MS). The active site environment of Plasmodium SHMT was probed based on changes in the fluorescence emission spectrum upon addition of amino acids and folate.ResultsAuto-induction media resulted in a two to three-fold higher yield of Pf- and PvSHMT (7.38 and 29.29 mg/L) compared to that produced in cells induced in LB-IPTG media. A convenient spectrophotometric activity assay coupling Plasmodium SHMT and MTHFD gave similar kinetic parameters to those previously obtained from the anaerobic assay coupling SHMT and 5,10-methylenetetrahydrofolate reductase (MTHFR); thus demonstrating the validity of the new assay procedure. The improved method was adopted to screen for Plasmodium SHMT inhibitors, of which some were originally designed as inhibitors of malarial dihydrofolate reductase. Plasmodium SHMT was slowly inactivated by thiosemicarbazide and formed a covalent intermediate, PLP-thiosemicarbazone.ConclusionsAuto-induction media offers a cost-effective method for the production of Plasmodium SHMTs and should be applicable for other Plasmodium enzymes. The SHMT-MTHFD coupled assay is equivalent to the SHMT-MTHFR coupled assay, but is more convenient for inhibitor screening and other studies of the enzyme. In addition to inhibitors of malarial SHMT, the development of species-specific, anti-SHMT inhibitors is plausible due to the presence of differential active sites on the Plasmodium enzymes.

Plasmodium serine hydroxymethyltransferase: indispensability and display of distinct localization

“…Although Pf cSHMT has been characterized in a number of reports [8,9,12,13,21], not much is known regarding the properties of putative Pf mSHMT, except for its mitochondrial location based on gene prediction and annotation in PlasmoDB. When the expression patterns of the two shmt forms were compared in P. falciparum during intra-erythrocytic developmental stages using semi-quantitative RT-PCR (normalized to that of Pfα-tubulin-2 expression), transcripts of both Pfshmt forms were detected throughout all erythrocytic stages, with peak transcript levels appearing during early and late trophozoite stage for putative Pfmshmt and Pfcshmt respectively (Figure 1).…”

“…Various classes of compounds, including 2,4-diaminopyrimidine, have been proposed to be effective inhibitors of Plasmodium SHMT based on binding affinity obtained from molecular docking calculations [34,35]. The recent study has shown that a number of 2,4-diaminopyrimidine compounds can inhibit Plasmodium SHMT [21]. Further optimization employing a target-based design approach should allow design of more effective anti-malarial drugs targeting Plasmodium SHMT.…”

Distinct biochemical properties of human serine hydroxymethyltransferase compared with the Plasmodium enzyme: implications for selective inhibition

“…The activity of hcSHMT was measured by following the protocol previously described . The coupled assay was performed under aerobic conditions in 50 m m HEPES buffer pH 7.0 containing 1 m m DTT, 0.5 m m EDTA, 2 m m l ‐serine, 250 μ m NADP + , 40 μ m THF, 10 μ m MTHFD and 0.05 μ m hcSHMT enzyme.…”

Kinetic Mechanism and the Rate-limiting Step of Plasmodium vivax Serine Hydroxymethyltransferase

“…The solution was allowed to cool, and the absorption of the resulting stable compound, 5,10-methenyltetrahydrofolate (CH (5). PvSHMT Preparation and Activity Assay-The expression and purification of recombinant PvSHMT were performed as previously reported (5,14). In brief, the expression plasmid containing pET17b-pvshmt was transformed into Escherichia coli BL21(DE3) and induced by the autoinduction method to express native PvSHMT without tag at 16°C in ZYP-5052-rich medium (5 mM Na 2 SO 4 , 2 mM MgSO 4 , 1ϫ NPS (25 mM Na 2 HPO 4 , 25 mM KH 2 PO 4 , 50 mM NH 4 Cl), 1ϫ 5052 (0.5%(w/v) glycerol, 0.05%(w/v) D-glucose, and 0.2%(w/v) ␣-lactose) containing 50 g/ml of ampicillin for overnight (ϳ16 -18 h).…”

“…The recombinant expression and purification of Plasmodium falciparum (PfSHMT) and Plasmodium vivax SHMT (PvSHMT) as well as their biochemical characterizations have been reported (5,(12)(13)(14). In general, both of these enzymes are similar in their enzymatic properties.…”