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

• Main Authors: Sopitthummakhun Kittipat, Thongpanchang Chawanee, Vilaivan Tirayut, Yuthavong Yongyuth, Chaiyen Pimchai, Leartsakulpanich Ubolsree
• Format: Article
• Language: English
• Published: BMC 2012-06-01
• Series: Malaria Journal
• Subjects: Serine hydroxymethyltransferase; <it>Plasmodium falciparum </it>; <it>Plasmodium vivax </it>; Pyridoxal-5-phosphate dependent enzyme; Thiosemicarbazide
• Online Access: http://www.malariajournal.com/content/11/1/194

<p><b>Abstract</b></p> <p><b>Background</b></p> <p>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. <it>Plasmodium</it> 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.</p> <p><b>Methods</b></p> <p>Production of recombinant <it>Plasmodium falciparum</it> SHMT (PfSHMT) and <it>Plasmodium vivax</it> 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. <it>Plasmodium</it> 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 <it>Plasmodium</it> SHMTs by thiosemicarbazide was investigated by spectrophotometry, high performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC-MS). The active site environment of <it>Plasmodium</it> SHMT was probed based on changes in the fluorescence emission spectrum upon addition of amino acids and folate.</p> <p><b>Results</b></p> <p>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 <it>Plasmodium</it> 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 <it>Plasmodium</it> SHMT inhibitors, of which some were originally designed as inhibitors of malarial dihydrofolate reductase. <it>Plasmodium</it> SHMT was slowly inactivated by thiosemicarbazide and formed a covalent intermediate, PLP-thiosemicarbazone.</p> <p><b>Conclusions</b></p> <p>Auto-induction media offers a cost-effective method for the production of <it>Plasmodium</it> SHMTs and should be applicable for other <it>Plasmodium</it> 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 <it>Plasmodium</it> enzymes.</p>