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...

Descripción completa

Detalles Bibliográficos
Autores principales: Sopitthummakhun, Kittipat, Thongpanchang, Chawanee, Vilaivan, Tirayut, Yuthavong, Yongyuth, Chaiyen, Pimchai, Leartsakulpanich, Ubolsree
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3502260/
https://www.ncbi.nlm.nih.gov/pubmed/22691309
http://dx.doi.org/10.1186/1475-2875-11-194
_version_ 1782250298955268096
author Sopitthummakhun, Kittipat
Thongpanchang, Chawanee
Vilaivan, Tirayut
Yuthavong, Yongyuth
Chaiyen, Pimchai
Leartsakulpanich, Ubolsree
author_facet Sopitthummakhun, Kittipat
Thongpanchang, Chawanee
Vilaivan, Tirayut
Yuthavong, Yongyuth
Chaiyen, Pimchai
Leartsakulpanich, Ubolsree
author_sort Sopitthummakhun, Kittipat
collection PubMed
description 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.
format Online
Article
Text
id pubmed-3502260
institution National Center for Biotechnology Information
language English
publishDate 2012
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-35022602012-11-21 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, Ubolsree Malar J Research 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. BioMed Central 2012-06-12 /pmc/articles/PMC3502260/ /pubmed/22691309 http://dx.doi.org/10.1186/1475-2875-11-194 Text en Copyright ©2012 Sopitthummakhun et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 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.
spellingShingle Research
Sopitthummakhun, Kittipat
Thongpanchang, Chawanee
Vilaivan, Tirayut
Yuthavong, Yongyuth
Chaiyen, Pimchai
Leartsakulpanich, Ubolsree
Plasmodium serine hydroxymethyltransferase as a potential anti-malarial target: inhibition studies using improved methods for enzyme production and assay
title Plasmodium serine hydroxymethyltransferase as a potential anti-malarial target: inhibition studies using improved methods for enzyme production and assay
title_full Plasmodium serine hydroxymethyltransferase as a potential anti-malarial target: inhibition studies using improved methods for enzyme production and assay
title_fullStr Plasmodium serine hydroxymethyltransferase as a potential anti-malarial target: inhibition studies using improved methods for enzyme production and assay
title_full_unstemmed Plasmodium serine hydroxymethyltransferase as a potential anti-malarial target: inhibition studies using improved methods for enzyme production and assay
title_short Plasmodium serine hydroxymethyltransferase as a potential anti-malarial target: inhibition studies using improved methods for enzyme production and assay
title_sort plasmodium serine hydroxymethyltransferase as a potential anti-malarial target: inhibition studies using improved methods for enzyme production and assay
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3502260/
https://www.ncbi.nlm.nih.gov/pubmed/22691309
http://dx.doi.org/10.1186/1475-2875-11-194
work_keys_str_mv AT sopitthummakhunkittipat plasmodiumserinehydroxymethyltransferaseasapotentialantimalarialtargetinhibitionstudiesusingimprovedmethodsforenzymeproductionandassay
AT thongpanchangchawanee plasmodiumserinehydroxymethyltransferaseasapotentialantimalarialtargetinhibitionstudiesusingimprovedmethodsforenzymeproductionandassay
AT vilaivantirayut plasmodiumserinehydroxymethyltransferaseasapotentialantimalarialtargetinhibitionstudiesusingimprovedmethodsforenzymeproductionandassay
AT yuthavongyongyuth plasmodiumserinehydroxymethyltransferaseasapotentialantimalarialtargetinhibitionstudiesusingimprovedmethodsforenzymeproductionandassay
AT chaiyenpimchai plasmodiumserinehydroxymethyltransferaseasapotentialantimalarialtargetinhibitionstudiesusingimprovedmethodsforenzymeproductionandassay
AT leartsakulpanichubolsree plasmodiumserinehydroxymethyltransferaseasapotentialantimalarialtargetinhibitionstudiesusingimprovedmethodsforenzymeproductionandassay