Abstract
BackgroundSerine hydroxymethyltransferase (SHMT), a pyridoxal phosphate-dependent enzyme, plays a vital role in the de novo pyrimidine biosynthesis pathway in malaria parasites. Two genes have been identified in Plasmodium spp. encoding a cytosolic SHMT (cSHMT) and putative mitochondria SHMT (mSHMT), but their roles have not been fully investigated.MethodsThe presence of Plasmodium SHMT isoforms in the intra-erythrocytic stage was assessed based on their gene expression using reverse transcription PCR (RT-PCR). Localization studies of Plasmodium SHMT isoforms were performed by transfection of fluorescent-tagged gene constructs into P. falciparum and expressions of fluorescent fusion proteins in parasites were observed using a laser scanning confocal microscope. Genetic targeting through homologous recombination was used to study the essentiality of SHMT in Plasmodium spp.ResultsSemi-quantitative RT-PCR revealed the expression of these two genes throughout intra-erythrocytic development. Localization studies using P. falciparum expressing fluorescent-tagged SHMT showed that PfcSHMT-red fluorescent fusion protein (PfcSHMT-DsRed) is localized in the cytoplasm, while PfmSHMT-green fluorescent fusion protein (PfmSHMT-GFP) co-localized with Mitotracker™-labelled mitochondria as predicted. The essentiality of plasmodial cSHMT was inferred from transfection experiments where recovery of viable knock-out parasites was not achieved, unless complemented with a functional equivalent copy of shmt.ConclusionsDistinct compartment localizations of PfSHMT were observed between cytoplasmic and mitochondrial isoforms, and evidence was provided for the indispensable role of plasmodial cSHMT indicating it as a valid target for development of novel anti-malarials.
Highlights
Serine hydroxymethyltransferase (SHMT), a pyridoxal phosphate-dependent enzyme, plays a vital role in the de novo pyrimidine biosynthesis pathway in malaria parasites
Expression pattern of PfSHMT PfcSHMT has been characterized in a number of reports [8,9,12,13,21], not much is known regarding the properties of putative PfmSHMT, 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 semiquantitative RT-PCR, transcripts of both Pfshmt forms were detected throughout all erythrocytic stages, with peak transcript levels appearing during early and late trophozoite stage for putative putative gene of mSHMT (Pfmshmt) and Pfcshmt respectively (Figure 1)
Summary
Serine hydroxymethyltransferase (SHMT), a pyridoxal phosphate-dependent enzyme, plays a vital role in the de novo pyrimidine biosynthesis pathway in malaria parasites. Two genes have been identified in Plasmodium spp. encoding a cytosolic SHMT (cSHMT) and putative mitochondria SHMT (mSHMT), but their roles have not been fully investigated. Based on DNA sequence search in PlasmoDB, there are two genes encoding SHMT in Plasmodium spp.: Plasmodium falciparum contains PFL1720w (PF3D7_1235600), a previously characterized cSHMT gene (Pfcshmt), and PF14_0534 (PF3D7_1456100), a putative gene of mSHMT (Pfmshmt). Comparison between the crystal structure of human cSHMT and homology model of PfcSHMT has revealed differences at the substrate binding site, which could be exploited for the development of specific anti-malarial inhibitors that do not cross inhibit the human enzymes [16]
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