Dataset Information

Catalytic and ligand-binding characteristics of Plasmodium falciparum serine hydroxymethyltransferase.

ABSTRACT: The plant-like, bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) from malaria parasites has been a good target for drug development. Dihydrofolate reductase (DHFR) is inhibited by clinically established antimalarials, pyrimethamine and cycloguanil. Thymidylate synthase (TS) is the target of potent experimental antimalarials such as 5-fluoroorotate and 1843U89. Another enzyme in folate recycling, serine hydroxymethyltransferase (SHMT), produces 5,10-methylenetetrahydrofolate which, in many cells, is required for the de novo, biosynthesis of thymidine and methionine. Thus, the biochemical characterization of malarial SHMT was of interest. The principle, active Plasmodium falciparum SHMT (PfSHMT) was expressed in E. coli and purified using an N-terminal histidine tag. Unlike the plant enzyme, but like the host enzyme, PfSHMT requires the cofactor pyridoxal 5'-phosphate for enzymatic activity. The substrate specificities for serine, tetrahydrofolate, and pyridoxal 5'-phosphate were comparable to those for SHMT from other organisms. Antifolates developed for DHFR and TS inhibited SHMT in the mid-micromolar range, offering insights into the binding preferences of SHMT but clearly leaving room for improved new inhibitors. As previously seen with P. falciparum DHFR-TS, PfSHMT bound its cognate mRNA but not control RNA for actin. RNA binding was not reversed with enzyme substrates. Unlike DHFR-TS, the SHMT RNA-protein interaction was not tight enough to inhibit translation. Another gene PF14_0534, previously proposed to code for an alternate mitochondrial SHMT, was also expressed in E. coli but found to be inactive. This protein, nor DHFR-TS, enhanced the catalytic activity of PfSHMT. The present results set the stage for developing specific, potent inhibitors of SHMT from P. falciparum.

SUBMITTER: Pang CK

PROVIDER: S-EPMC2741015 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Catalytic and ligand-binding characteristics of Plasmodium falciparum serine hydroxymethyltransferase.

Pang Cullen K T CK Hunter Joshua H JH Gujjar Ramesh R Podutoori Ramulu R Bowman Julie J Mudeppa Devaraja G DG Rathod Pradipsinh K PK

Molecular and biochemical parasitology 20090708 1

The plant-like, bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) from malaria parasites has been a good target for drug development. Dihydrofolate reductase (DHFR) is inhibited by clinically established antimalarials, pyrimethamine and cycloguanil. Thymidylate synthase (TS) is the target of potent experimental antimalarials such as 5-fluoroorotate and 1843U89. Another enzyme in folate recycling, serine hydroxymethyltransferase (SHMT), produces 5,10-methylenetetrahydrofolate wh ...[more]

PMID: 19591883

Similar Datasets

Project description:BackgroundThe folate pathway enzyme serine hydroxymethyltransferase (SHMT) converts serine to glycine and 5,10-methylenetetrahydrofolate and is essential for the acquisition of one-carbon units for subsequent transfer reactions. 5,10-methylenetetrahydrofolate is used by thymidylate synthase to convert dUMP to dTMP for DNA synthesis. In Plasmodium falciparum an enzymatically functional SHMT (PfSHMTc) and a related, apparently inactive isoform (PfSHMTm) are found, encoded by different genes. Here, patterns of localization of the two isoforms during the parasite erythrocytic cycle are investigated.MethodsPolyclonal antibodies were raised to PfSHMTc and PfSHMTm, and, together with specific markers for the mitochondrion and apicoplast, were employed in quantitative confocal fluorescence microscopy of blood-stage parasites.ResultsAs well as the expected cytoplasmic occupancy of PfSHMTc during all stages, localization into the mitochondrion and apicoplast occurred in a stage-specific manner. Although early trophozoites lacked visible organellar PfSHMTc, a significant percentage of parasites showed such fluorescence during the mid-to-late trophozoite and schizont stages. In the case of the mitochondrion, the majority of parasites in these stages at any given time showed no marked PfSHMTc fluorescence, suggesting that its occupancy of this organelle is of limited duration. PfSHMTm showed a distinctly more pronounced mitochondrial location through most of the erythrocytic cycle and GFP-tagging of its N-terminal region confirmed the predicted presence of a mitochondrial signal sequence. Within the apicoplast, a majority of mitotic schizonts showed a marked concentration of PfSHMTc, whose localization in this organelle was less restricted than for the mitochondrion and persisted from the late trophozoite to the post-mitotic stages. PfSHMTm showed a broadly similar distribution across the cycle, but with a distinctive punctate accumulation towards the ends of elongating apicoplasts. In very late post-mitotic schizonts, both PfSHMTc and PfSHMTm were concentrated in the central region of the parasite that becomes the residual body on erythrocyte lysis and merozoite release.ConclusionsBoth PfSHMTc and PfSHMTm show dynamic, stage-dependent localization among the different compartments of the parasite and sequence analysis suggests they may also reversibly associate with each other, a factor that may be critical to folate cofactor function, given the apparent lack of enzymic activity of PfSHMTm.

Dataset Information

Catalytic and ligand-binding characteristics of Plasmodium falciparum serine hydroxymethyltransferase.

Publications

Catalytic and ligand-binding characteristics of Plasmodium falciparum serine hydroxymethyltransferase.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets