Arabidopsis SHMT1, a serine hydroxymethyltransferase that functions in the photorespiratory pathway influences resistance to biotic and abiotic stress

We found that a recessive mutation, shmt1-1 , causes aberrant regulation of cell death resulting in chlorotic and necrotic lesion formation under a variety of environmental conditions. Salicylic acid-inducible genes and genes involved in H₂O₂ detoxification were expressed constitutively in shmt1-1 plants in direct correlation with the severity of the lesions. The shmt1-1 mutants were more susceptible than control plants to infection with biotrophic and necrotrophic pathogens, developing severe infection symptoms in a high percentage of infected leaves. In addition, mutants carrying shmt1-1 or a loss-of-function shmt1-2 allele, were smaller and showed a greater loss of chlorophyll and greater accumulation of H₂O₂ than wild-type plants when subjected to salt stress. SHMT1 was map-based cloned and found to encode a serine hydroxymetyltransferase (SHMT1) involved in the photorespiratory pathway. Our results indicate that this enzymatic activity plays a critical role in controlling the cell damage provoked by abiotic stresses such as high light and salt and in restricting pathogen-induced cell death, supporting the notion that photorespiration forms part of the dissipatory mechanisms of plants to minimize production of reactive oxygen species (ROS) at the chloroplast and to mitigate oxidative damage. Moreover, results shown here indicate that whereas production of ROS is an essential component of the hypersensitive defense response, the excessive accumulation of these toxic compounds impairs cell death containment and counteracts the effectiveness of the plant defenses to restrict pathogen infection.     

Role of methionine in the regulation of serine hydroxymethyltransferase in Eschericia coli.

Significant derepression of serine hydroxymethyltransferase is observed when metE or metF mutants of Escherichia coli K-12 are grown on D-methionine sulfoxide instead of L-methionine. The derepression is not prevented by addition of glycine, adenosine, guanosine, guanosine, and thymidine to the growth medium of methionine-limited metF cells showing that the effect is not due to a secondary deficiency of these nutrients. On the other hand, methionine-limited growth of a metA mutant leads to derepression of met regulon enzymes, but only a marginal increase in serine hydroxymethyltransferase activity. A prototrophic metJ strain grown on minimal medium has about the same serine hydroxymethyltransferase as the wild type. The enzyme activity of the metJ strain is not influenced by methionine, but it is partially repressed by glycine, adenosine, and thymidine. metK strains have about twice as much serine hydroxymethyltransferase activity as wild-type cells when grown on minimal medium; but when both types of cells are grown on medium supplemented with glycine, adenosine, guanosine, and thymidine, their enzyme activities are about the same. The results show that methionine limitation can lead to depression of serine hydroxymethyltransferase, but that the regulatory system is different from the one which controls the methionine regulon.     

The serine hydroxymethyltransferase of Plasmodium lophurae.

Plasmodium lophurae serine hydroxymethyltransferase (EC 2.1.2.1) was partially purified and characterized by (NH4)2SO4 fractionation and chromatography on Sephadex G-100. The enzyme, precipitated by 3.0.3.3 M (NH4)2SO4, had a molecular weight of 68,300 as estimated by exclusion chromatography on G-100. The pH optimum of the enzyme was 6.8-7.6 in sodium phosphate-citrate buffer. Citrate stabilized the enzyme during storage in phosphate buffer at 4 C. The Km was 4.3 X 10(-3) M for L-serine and 2.5 X 10(-4) M for tetrahydrofolate.     

Two anthranilate synthase genes in Arabidopsis: defense-related regulation of the tryptophan pathway.

Arabidopsis thaliana has two genes, ASA1 and ASA2, encoding the alpha subunit of anthranilate synthase, the enzyme catalyzing the first reaction in the tryptophan biosynthetic pathway. As a branchpoint enzyme in aromatic amino acid biosynthesis, anthranilate synthase has an important regulatory role. The sequences of the plant genes are homologous to their microbial counterparts. Both predicted proteins have putative chloroplast transit peptides at their amino termini and conserved amino acids involved in feedback inhibition by tryptophan. ASA1 and ASA2 cDNAs complement anthranilate synthase alpha subunit mutations in the yeast Saccharomyces cerevisiae and in Escherichia coli, confirming that both genes encode functional anthranilate synthase proteins. The distributions of ASA1 and ASA2 mRNAs in various parts of Arabidopsis plants are overlapping but nonidentical, and ASA1 mRNA is approximately 10 times more abundant in whole plants. Whereas ASA2 is expressed at a constitutive basal level, ASA1 is induced by wounding and bacterial pathogen infiltration, suggesting a novel role for ASA1 in the production of tryptophan pathway metabolites as part of an Arabidopsis defense response. Regulation of key steps in aromatic amino acid biosynthesis in Arabidopsis appears to involve differential expression of duplicated genes.     

Organization of two genes encoding cytotoxic T lymphocyte-specific serine proteases CCPI and CCPII

The genes encoding two recently described cytotoxic T cell proteases, CCPI and CCPII, have been isolated and sequenced. The organizations of the coding and noncoding portions of the two genes are very similar to each other and also to the gene encoding rat mast cell protease type II. Similarly to other serine protease genes, each of the active-site residues is contained on a separate exon; however, two introns were found in particularly interesting positions. One occurs within the postulated activation dipeptide and the other in a position close to the active-site Asp residue. This latter intron interrupts the amino acid sequence in the invariant core region of the protein. We believe that these genes represent a new subfamily of serine protease genes.     

Haploinsufficiency of cytosolic serine hydroxymethyltransferase in the Smith-Magenis syndrome.

Folate-dependent one-carbon metabolism is critical for the synthesis of numerous cellular constituents required for cell growth, and serine hydroxymethyltransferase (SHMT) is central to this process. Our studies reveal that the gene for cytosolic SHMT (cSHMT) maps to the critical interval for Smith-Magenis syndrome (SMS) on chromosome 17p11.2. The basic organization of the cSHMT locus on chromosome 17 was determined and was found to be deleted in all 26 SMS patients examined by PCR, FISH, and/or Southern analysis. Furthermore, with respect to haploinsufficiency, cSHMT enzyme activity in patient lymphoblasts was determined to be approximately 50% that of unaffected parent lymphoblasts. Serine, glycine, and folate levels were also assessed in three SMS patients and were found to be within normal ranges. The possible effects of cSHMT hemizygosity on the SMS phenotype are discussed.     

The presequence of Arabidopsis serine hydroxymethyltransferase SHM2 selectively prevents import into mesophyll mitochondria

Serine hydroxymethyltransferases (SHMs) are important enzymes of cellular one-carbon metabolism and are essential for the photorespiratory glycine-into-serine conversion in leaf mesophyll mitochondria. In Arabidopsis (Arabidopsis thaliana), SHM1 has been identified as the photorespiratory isozyme, but little is known about the very similar SHM2. Although the mitochondrial location of SHM2 can be predicted, some data suggest that this particular isozyme could be inactive or not targeted into mitochondria. We report that SHM2 is a functional mitochondrial SHM. In leaves, the presequence of SHM2 selectively hinders targeting of the enzyme into mesophyll mitochondria. For this reason, the enzyme is confined to the vascular tissue of wild-type Arabidopsis, likely the protoxylem and/or adjacent cells, where it occurs together with SHM1. The resulting exclusion of SHM2 from the photorespiratory environment of mesophyll mitochondria explains why this enzyme cannot substitute for SHM1 in photorespiratory metabolism. Unlike the individual shm1 and shm2 null mutants, which require CO(2)-enriched air to inhibit photorespiration (shm1) or do not show any visible impairment (shm2), double-null mutants cannot survive in CO(2)-enriched air. It seems that SHM1 and SHM2 operate in a redundant manner in one-carbon metabolism of nonphotorespiring cells with a high demand of one-carbon units; for example, during lignification of vascular cells. We hypothesize that yet unknown kinetic properties of SHM2 might render this enzyme unsuitable for the high-folate conditions of photorespiring mesophyll mitochondria.     

Subcellular distribution of serine hydroxymethyltransferase in Plasmodium lophurae.

Subcellular fractions of the bird malaria, $\text{Plasmodium lophurae}$ were prepared by differential centrifugation. Cytochrome oxidase activity was located in the mitochondrial fraction. A major portion of glutamate dehydrogenase activity was found in the mitochondrial fraction with the remainder in the ribosomal and cytosolic fractions. Malate dehydrogenase and serine hydroxymethyltransferase activities were located primarily in the cytosolic fraction.     

The organisation and expression of the genes encoding the mitochondrial glycine decarboxylase complex and serine hydroxymethyltransferase in pea (Pisum sativum)

Summary Restriction fragment length polymorphisms have been used to determine the chromosomal location of the genes encoding the glycine decarboxylase complex (GDC) and serine hydroxymethyltransferase (SHMT) of pea leaf mitochondria. The genes encoding the H subunit of GDC and the genes encoding SHMT both show linkage to the classical group I marker i. In addition, the genes for the P protein of GDC show linkage to the classic group I marker a. The genes for the L and T proteins of GDC are linked to one another and are probably situated on the satellite of chromosome 7. The mRNAs encoding the five polypeptides that make up GDC and SHMT are strongly induced when dark-grown etiolated pea seedlings are placed in the light. Similarly, when mature plants are placed in the dark for 48 h, the levels of both GDC protein and SHMT mRNAs decline dramatically and then are induced strongly when these plants are returned to the light. During both treatments a similar pattern of mRNA induction is observed, with the mRNA encoding the P protein of GDC being the most rapidly induced and the mRNA for the H protein the slowest. Whereas during the greening of etiolated seedlings the polypeptides of GDC and SHMT show patterns of accumulation similar to those of the corresponding mRNAs, very little change in the level of the polypeptides is seen when mature plants are placed in the dark and then re-exposed to the light.     

Purification and properties of serine hydroxymethyltransferase from Sulfolobus solfataricus.

Serine hydroxymethyltransferase (SHMT) catalyzes the reversible cleavage of serine to glycine with the transfer of the one-carbon group to tetrahydrofolate to form 5,10-methylenetetrahydrofolate. No SHMT has been purified from a nonmethanogenic Archaea strain, in part because this group of organisms uses modified folates as the one-carbon acceptor. These modified folates are not readily available for use in assays for SHMT activity. This report describes the purification and characterization of SHMT from the thermophilic organism Sulfolobus solfataricus. The exchange of the alpha-proton of glycine with solvent protons in the absence of the modified folate was used as the activity assay. The purified protein catalyzes the synthesis of serine from glycine and a synthetic derivative of a fragment of the natural modified folate found in S. solfataricus. Replacement of the modified folate with tetrahydrofolate did not support serine synthesis. In addition, this SHMT also catalyzed the cleavage of both allo-threonine and beta-phenylserine in the absence of the modified folate. The cleavage of these two amino acids in the absence of tetrahydrofolate is a property of other characterized SHMTs. The enzyme contains covalently bound pyridoxal phosphate. Sequences of three peptides showed significant similarity with those of peptides of SHMTs from two methanogens.     

Identification of active-site residues of sheep liver serine hydroxymethyltransferase.

Chemical modification of amino acid residues with phenylglyoxal, N-ethylmaleimide and diethyl pyrocarbonate indicated that at least one residue each of arginine, cysteine and histidine were essential for the activity of sheep liver serine hydroxymethyltransferase. The second-order rate constants for inactivation were calculated to be 0.016 mM-1 X min-1 for phenylglyoxal, 0.52 mM-1 X min-1 for N-ethylmaleimide and 0.06 mM-1 X min-1 for diethyl pyrocarbonate. Different rates of modification of these residues in the presence and in the absence of substrates and the cofactor pyridoxal 5'-phosphate as well as the spectra of the modified protein suggested that these residues might occur at the active site of the enzyme.     

Regulation of monkey liver serine hydroxymethyltransferase by nicotinamide nucleotide.

The positive homotropic binding of tetrahydrofolate to monkey liver serine hydroxymethyltransferase was abolished on preincubating the enzyme with NADH and NADPH. NAD+ was a negative heterotropic effector, whereas NADP+ was without effect. The allosteric effects of nicotinamide nucleotides on the serine hydroxymethyltransferase, reported for the first time, lead to a better understanding of the regulation of the metabolic interconversion of folate coenzymes.