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


METHODS
Production 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.


RESULTS
Auto-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.


CONCLUSIONS
Auto-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.
Department of Biochemistry and Center of Excellence in Protein Structure & Function, Faculty of Science, Mahidol University, Rama 6 Road Bangkok 10400, Thailand.
Kittipat
K
Sopitthummakhun
Chawanee
C
Thongpanchang
Tirayut
T
Vilaivan
Yongyuth
Y
Yuthavong
Pimchai
P
Chaiyen
Ubolsree
U
Leartsakulpanich
D000962:Antimalarials / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D012696:Glycine Hydroxymethyltransferase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP) / Q000378:metabolism; D010963:Plasmodium falciparum / Q000201:enzymology*; D010966:Plasmodium vivax / Q000201:enzymology*; D013053:Spectrophotometry / Q000379:methods
D000962:Antimalarials; D004791:Enzyme Inhibitors; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP); D012696:Glycine Hydroxymethyltransferase