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Resolution and characterization of the glycine-cleavage reaction in pea leaf mitochondria. Properties of the forward reaction catalysed by glycine decarboxylase and serine hydroxymethyltransferase.

ABSTRACT: High-molecular-mass proteins from pea (Pisum sativum) mitochondrial matrix retained on an XM-300 Diaflo membrane ('matrix extract') exhibited high rates of glycine oxidation in the presence of NAD+ and tetrahydropteroyl-L-glutamic acid (H4 folate) as long as the medium exhibited a low ionic strength. Serine hydroxymethyltransferase (SHMT) (4 x 53 kDa) and the four proteins of the glycine-cleavage system, including a pyridoxal phosphate-containing enzyme ('P-protein'; 2 x 97 kDa), a carrier protein containing covalently bound lipoic acid ('H-protein'; 15.5 kDa), a protein exhibiting lipoamide dehydrogenase activity ('L-protein'; 2 x 61 kDa) and an H4 folate-dependent enzyme ('T-protein'; 45 kDa) have been purified to apparent homogeneity from the matrix extract by using gel filtration, ion-exchange and phenyl-Superose fast protein liquid chromatography. Gel filtration on Sephacryl S-300 in the presence of 50 mM-KCl proved to be the key step in disrupting this complex. During the course of glycine oxidation catalysed by the matrix extract a steady-state equilibrium in the production and utilization of 5,10-methylene-H4 folate was reached, suggesting that glycine cleavage and SHMT are linked together via a soluble pool of H4 folate. The rate of glycine oxidation catalysed by the matrix extract was sensitive to the NADH/NAD+ molar ratios, because NADH competitively inhibited the reaction catalysed by lipoamide dehydrogenase.

SUBMITTER: Bourguignon J

PROVIDER: S-EPMC1135205 | biostudies-other | 1988 Oct

REPOSITORIES: biostudies-other

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Interaction between glycine decarboxylase, serine hydroxymethyltransferase and tetrahydrofolate polyglutamates in pea leaf mitochondria.

Project description:The aim of the present work was to further determine how the T-protein of the glycine-cleavage system and serine hydroxy-methyltransferase (SHMT), two folate-dependent enzymes from pea leaf mitochondria, interact through a common pool of tetrahydrofolate polyglutamates (H4PteGlun). It was observed that the binding affinity of tetrahydrofolate polyglutamates for these proteins continuously increased with increasing number of glutamates up to six residues. It was also established that, once bound to the proteins, tetrahydrofolate, a very O2-sensitive molecule, was protected from oxidative degradation. The dissociation constants (Kd) of H4PteGlu5, the most predominant form of polyglutamate in the mitochondria, were approximately 0.5 microM for both T-protein and SHMT, whereas the Kd values of CH2-H4PteGlu5 were higher, 2.7 and 7 microM respectively. In a matrix extract from pea leaf mitochondria, the maximal activity of the glycine-cleavage system was about 2.5 times higher than the maximal activity of SHMT. This resulted in a permanent disequilibrium of the SHMT-catalysed reaction which was therefore driven toward the production of serine and H4PteGlun, the thermodynamically unfavourable direction. Indeed, measurements of the steady-state ratio of CH2-H4PteGlun/H4PteGlun (n = 1 or n = 5) during the course of glycine oxidation demonstrated that the methylene form accounted for 65-80% of the folate pool. This indicates that, in our in vitro experiments, CH2-H4PteGlun with long polyglutamate chains accumulated in the bulk medium. This observation suggests that, in these in vitro experiments at least, there was no channelling of CH2-H4PteGlu5 between the T-protein and SHMT.

| S-EPMC1137213 | biostudies-other

Effects of tetrahydrofolate polyglutamates on the kinetic parameters of serine hydroxymethyltransferase and glycine decarboxylase from pea leaf mitochondria.

Project description:Plant tissues contain highly conjugated forms of folate. Despite this, the ability of plant folate-dependent enzymes to utilize tetrahydrofolate polyglutamates has not been examined in detail. In leaf mitochondria, the glycine-cleavage system and serine hydroxymethyltransferase, present in large amounts in the matrix space and involved in the photorespiratory cycle, necessitate the presence of tetrahydrofolate as a cofactor. The aim of the present work was to determine whether glutamate chain length (one to six glutamate residues) influenced the affinity constant for tetrahydrofolate and the maximal velocities displayed by these two enzymes. The results show that the affinity constant decreased by at least one order of magnitude when the tetrahydrofolate substrate contained three or more glutamate residues. In contrast, maximal velocities were not altered in the presence of these substrates. These results are consistent with analyses of mitochondrial folates which revealed a pool of polyglutamates dominated by tetra and pentaglutamates. The equilibrium constant of the serine hydroxymethyltransferase suggests that, during photorespiration, the reaction must be permanently pushed toward the formation of serine (the unfavourable direction) to allow the recycling of tetrahydrofolate necessary for the operation of the glycine decarboxylase T-protein.

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cDNA cloning, primary structure and gene expression for H-protein, a component of the glycine-cleavage system (glycine decarboxylase) of pea (Pisum sativum) leaf mitochondria.

Project description:We have isolated and characterized cDNA clones encoding the H-protein of the glycine-cleavage system of pea (Pisum sativum) leaf mitochondria. The deduced primary structure revealed that the 131-amino-acid polypeptide is cytoplasmically synthesized with a 34-amino-acid mitochondrial targeting peptide. The lipoate-binding site was assigned to be lysine-63, as deduced from a sequence comparison with several lipoate-bearing proteins. The expression of the gene encoding H-protein was shown to occur specifically in the leaf tissue, with light exerting an additional effect by increasing the mRNA levels severalfold. Two polyadenylation sites were found in the mRNA, and a single-copy gene encoding the H-protein was detected in pea genome.

| S-EPMC1131509 | biostudies-other

Design strategy for serine hydroxymethyltransferase probes based on retro-aldol-type reaction.

Project description:Serine hydroxymethyltransferase (SHMT) is an enzyme that catalyzes the reaction that converts serine to glycine. It plays an important role in one-carbon metabolism. Recently, SHMT has been shown to be associated with various diseases. Therefore, SHMT has attracted attention as a biomarker and drug target. However, the development of molecular probes responsive to SHMT has not yet been realized. This is because SHMT catalyzes an essential yet simple reaction; thus, the substrates that can be accepted into the active site of SHMT are limited. Here, we focus on the SHMT-catalyzed retro-aldol reaction rather than the canonical serine-glycine conversion and succeed in developing fluorescent and 19F NMR molecular probes. Taking advantage of the facile and direct detection of SHMT, the developed fluorescent probe is used in the high-throughput screening for human SHMT inhibitors, and two hit compounds are obtained.

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Structure-based mechanism for early PLP-mediated steps of rabbit cytosolic serine hydroxymethyltransferase reaction.

Project description:Serine hydroxymethyltransferase catalyzes the reversible interconversion of L-serine and glycine with transfer of one-carbon groups to and from tetrahydrofolate. Active site residue Thr254 is known to be involved in the transaldimination reaction, a crucial step in the catalytic mechanism of all pyridoxal 5'-phosphate- (PLP-) dependent enzymes, which determines binding of substrates and release of products. In order to better understand the role of Thr254, we have expressed, characterized, and determined the crystal structures of rabbit cytosolic serine hydroxymethyltransferase T254A and T254C mutant forms, in the absence and presence of substrates. These mutants accumulate a kinetically stable gem-diamine intermediate, and their crystal structures show differences in the active site with respect to wild type. The kinetic and crystallographic data acquired with mutant enzymes permit us to infer that conversion of gem-diamine to external aldimine is significantly slowed because intermediates are trapped into an anomalous position by a misorientation of the PLP ring, and a new energy barrier hampers the transaldimination reaction. This barrier likely arises from the loss of the stabilizing hydrogen bond between the hydroxymethyl group of Thr254 and the ? -amino group of active site Lys257, which stabilizes the external aldimine intermediate in wild type SHMTs.

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Stoichiometry of two plant glycine decarboxylase complexes and comparison with a cyanobacterial glycine cleavage system

Project description:The multienzyme glycine cleavage system (GCS) converts glycine and tetrahydrofolate to the one-carbon compound 5,10-methylenetetrahydrofolate, which is of vital importance for most if not all organisms. Photorespiring plant mitochondria contain very high levels of GCS proteins organized as a fragile glycine decarboxylase complex (GDC). The aim of this study is to provide mass spectrometry-based stoichiometric data for the plant leaf GDC and examine whether complex formation could be a general property of the GCS in photosynthesizing organisms. To this end, we investigated the mitochondrial content, the stoichiometry and the isoprotein composition of the Arabidopsis thaliana leaf GDC in comparison with that of Pisum sativum using stable-isotope-labeled peptides (QconCAT and synthetic labeled peptides) and compared the results to label-free, Hi3 peptide quantification.

2020-04-23 | PXD018211 | Pride

Glycine decarboxylase and pyruvate dehydrogenase complexes share the same dihydrolipoamide dehydrogenase in pea leaf mitochondria: evidence from mass spectrometry and primary-structure analysis.

Project description:In order to compare the dihydrolipoamide dehydrogenase associated with the pyruvate dehydrogenase complex (E3) with that associated with the glycine decarboxylase complex (L-protein), we report for the first time the purification and characterization of the E3 component from pea leaf mitochondria. The first 30 amino acids of the N-terminal sequence of the mature E3 protein are identical with those of the mature L-protein of the glycine decarboxylase complex. Electrospray ionization-mass spectrometric analysis of E3 and the L-protein gave exactly the same molecular mass of 49,753 +/- 5 Da. We have also confirmed the primary structure of the L-protein, in particular the C-terminal sequence, deduced from the cDNA published by Bourguignon, Macherel, Neuburger and Douce [(1992) Eur. J. Biochem. 204, 865-873]. Western-blot analysis shows that specific polyclonal antibodies raised against the L-protein recognize specifically both E3 and L-protein but not the porcine dihydrolipoamide dehydrogenase. We conclude that, in pea leaf mitochondria, the pyruvate dehydrogenase and glycine decarboxylase complexes share the same dihydrolipoamide dehydrogenase. We have also confirmed by MS analysis that the FAD is not covalently bound to the enzyme.

| S-EPMC1216887 | biostudies-other

A comparative study of the kinetics and stereochemistry of the serine hydroxymethyltransferase- and tryptophan synthase-catalysed exchange of the pro-2R and pro-2S protons of glycine.

Project description:The stereospecificity of the serine hydroxymethyltransferase (EC 2.1.2.1)- and tryptophan synthase (EC 4.2.1.20)- catalysed exchange of the pro-2R and pro-2S alpha-protons of glycine was investigated by using 13C n.m.r. The exchange process is described in terms of a minimal four-step mechanism, and a method for analysing the exchange process by complete progress curves is presented. It is shown that serine hydroxymethyltransferase does not have absolute stereospecificity for the pro-2S-proton of glycine, but it catalyses the exchange of this proton 7400 times faster than the pro-2R proton of glycine. Tryptophan synthase is shown preferentially to catalyse the exchange of the pro-2R proton of glycine at a rate 380 times faster than the pro-2S proton of glycine. The exchange rates for the rapidly exchanged alpha-protons of glycine are similar for both enzymes. However, the exchange rates of the slowly exchanged alpha-protons differ by an order of magnitude. The structural features that may be responsible for the differences in the stereospecificity of the two enzymes are discussed.

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Plasmodium serine hydroxymethyltransferase: indispensability and display of distinct localization.

Project description:BACKGROUND: 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. METHODS: The 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. RESULTS: Semi-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. CONCLUSIONS: Distinct 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.

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Disruption of the SHM2 gene, encoding one of two serine hydroxymethyltransferase isoenzymes, reduces the flux from glycine to serine in Ashbya gossypii.

Project description:Riboflavin overproduction in the ascomycete Ashbya gossypii is limited by glycine, a precursor of purine biosynthesis, and therefore an indicator of glycine metabolism. Disruption of the SHM 2 gene, encoding a serine hydroxymethyltransferase, resulted in a significant increase in riboflavin productivity. Determination of the enzyme's specific activity revealed a reduction from 3 m-units/mg of protein to 0.5 m-unit/mg protein. The remaining activity was due to an isoenzyme encoded by SHM 1, which is probably mitochondrial. A hypothesis proposed to account for the enhanced riboflavin overproduction of SHM 2-disrupted mutants was that the flux from glycine to serine was reduced, thus leading to an elevated supply with the riboflavin precursor glycine. Evidence for the correctness of that hypothesis was obtained from (13)C-labelling experiments. When 500 microM 99% [1-(13)C]threonine was fed, more than 50% of the label was detected in C-1 of glycine resulting from threonine aldolase activity. More than 30% labelling determined in C-1 of serine can be explained by serine synthesis via serine hydroxymethyltransferase. Knockout of SHM 1 had no detectable effect on serine labelling, but disruption of SHM 2 led to a decrease in serine (2-5%) and an increase in glycine (59-67%) labelling, indicating a changed carbon flux.

| S-EPMC1223077 | biostudies-other

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