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

Sopitthummakhun Kittipat, Thongpanchang Chawanee, Vilaivan Tirayut, Yuthavong Yongyuth, Chaiyen Pimchai, Leartsakulpanich, Leartsakulpanich Ubolsree

Description

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 .

Sujets

• Serine hydroxymethyltransferase
• Plasmodium falciparum
• Plasmodium vivax
• Semicarbazide

Informations

• Publié par: biomed
• Publié le: 01 janvier 2012
• Nombre de lectures: 5
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Sopitthummakhunet al. Malaria Journal2012,11:194 http://www.malariajournal.com/content/11/1/194

R E S E A R C HOpen Access Plasmodiumserine hydroxymethyltransferase as a potential antimalarial target: inhibition studies using improved methods for enzyme production and assay 1 23 2 Kittipat Sopitthummakhun , Chawanee Thongpanchang , Tirayut Vilaivan , Yongyuth Yuthavong , 1* 2* Pimchai Chaiyenand Ubolsree Leartsakulpanich

Abstract 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.Plasmodiumserine 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 recombinantPlasmodium falciparumSHMT (PfSHMT) andPlasmodium vivaxSHMT (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.PlasmodiumSHMT 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 ofPlasmodiumSHMTs by thiosemicarbazide was investigated by spectrophotometry, high performance liquid chromatography (HPLC), and liquid chromatographymass spectrometry (LCMS). The active site environment of PlasmodiumSHMT 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 couplingPlasmodiumSHMT 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 forPlasmodiumSHMT inhibitors, of which some were originally designed as inhibitors of malarial dihydrofolate reductase.PlasmodiumSHMT was slowly inactivated by thiosemicarbazide and formed a covalent intermediate, PLPthiosemicarbazone. Conclusions:Autoinduction media offers a costeffective method for the production ofPlasmodiumSHMTs and should be applicable for otherPlasmodiumenzymes. 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

* Correspondence: [e-mail]; [e-mail] 1 Department of Biochemistry and Center of Excellence in Protein Structure & Function, Faculty of Science, Mahidol University, Rama 6 Road Bangkok 10400, Thailand 2 National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Paholyothin Road, Pathumthani 12120, Thailand Full list of author information is available at the end of the article

© 2012 Sopitthummakhun et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.