Serine Hydroxymethyltransferase Activity Research Articles

Cloning, DNA shuffling and expression of serine hydroxymethyltransferase gene from Escherichia coli strain AB90054

A gene ( glyA) encoding serine hydroxymethyltransferase from Escherichia coli strain AB90054 was cloned, sequenced and expressed in E. coli. The gene consists of 1254 bp and encodes a protein of 417 amino acids. Model-free approaches (error-prone PCR to introduce random mutations, DNA shuffling to combine positive mutations, and high throughput screening of the resultant mutant libraries) have been used to enhance the catalytic activity of serine hydroxymethyltransferase. After three rounds of error-prone PCR and three sequential generations of DNA shuffling were performed on the glyA gene and recombinants were screened using a genetic complement coupled assay, a mutant 3E7 with approximately 8-fold increased enzyme activity and 41-fold increased enzyme productivity compared with its wild-type parent was obtained. Mutant 3E7 contains eight amino acid substitutions. The purified 3E7 and wild-type serine hydroxymethyltransferase each showed a single band at about 45 kDa by SDS-polyacrylamide gel electrophoresis. Some properties of the purified WT enzyme and evolved enzyme were also determined in this work. Homology model structure of 3E7 indicated that all the mutations do not interact directly with substrate but S355N exert its effect by forming H-bonds with THF and contributing to transition state stabilization.

The photorespiratory Arabidopsis shm1 mutant is deficient in SHM1.

Mitochondrial serine hydroxymethyltransferase (SHMT), combined with glycine decarboxylase, catalyzes an essential sequence of the photorespiratory C2 cycle, namely, the conversion of two molecules of glycine into one molecule each of CO2, NH4+, and serine. The Arabidopsis (Arabidopsis thaliana) mutant shm (now designated shm1-1) is defective in mitochondrial SHMT activity and displays a lethal photorespiratory phenotype when grown at ambient CO2, but is virtually unaffected at elevated CO2. The Arabidopsis genome harbors seven putative SHM genes, two of which (SHM1 and SHM2) feature predicted mitochondrial targeting signals. We have mapped shm1-1 to the position of the SHM1 gene (At4g37930). The mutation is due to a G --> A transition at the 5' splice site of intron 6 of SHM1, causing aberrant splicing and a premature termination of translation. A T-DNA insertion allele of SHM1, shm1-2, and the F1 progeny of a genetic cross between shm1-1 and shm1-2 displayed the same conditional lethal phenotype as shm1-1. Expression of wild-type SHM1 under the control of either the cauliflower mosaic virus 35S or the SHM1 promoter in shm1-1 abrogated the photorespiratory phenotype of the shm mutant, whereas overexpression of SHM2 or expression of SHM1 under the control of the SHM2 promoter did not rescue the mutant phenotype. Promoter-beta-glucuronidase analyses revealed that SHM1 is predominantly expressed in leaves, whereas SHM2 is mainly transcribed in the shoot apical meristem and roots. Our findings establish SHM1 as the defective gene in the Arabidopsis shm1-1 mutant.

Antisense reduction of serine hydroxymethyltransferase results in diurnal displacement of NH4+ assimilation in leaves of Solanum tuberosum

Serine hydroxymethyltransferase (SHMT) is part of the mitochondrial enzyme complex catalysing the photorespiratory production of serine, ammonium and CO(2) from glycine. Potato plants (Solanum tuberosum cv. Solara) with antisensed SHMT were generated to investigate whether photorespiratory intermediates accumulated during light lead to nocturnal activation of the nitrogen-assimilating enzymes glutamine synthetase (GS) and glutamate synthase (GOGAT). The transformant lines contained 70-90% less SHMT protein, and exhibited a corresponding decrease in mitochondrial SHMT activity. SHMT antisense plants displayed lower photosynthetic capacity and accumulated glycine in light. Glycine was converted to serine in the second half of the light period, while serine, ammonium and glutamine showed an inverse diurnal rhythm and reached highest values in darkness. GS/GOGAT protein levels and activities in the transgenics also reached maximum levels in darkness. The diurnal displacement of NH(4)(+) assimilation was accompanied by a change in the subunit composition of GS(2), but not GS(1). It is concluded that internal accumulation of post-photorespiratory ammonium is leading to nocturnal activation of GS/GOGAT, and that the time shift in ammonia assimilation can constitute part of a strategy to survive photorespiratory impairment.

5-Formyltetrahydrofolate Is an Inhibitory but Well Tolerated Metabolite in Arabidopsis Leaves

5-Formyltetrahydrofolate (5-CHO-THF) is formed via a second catalytic activity of serine hydroxymethyltransferase (SHMT) and strongly inhibits SHMT and other folate-dependent enzymes in vitro. The only enzyme known to metabolize 5-CHO-THF is 5-CHO-THF cycloligase (5-FCL), which catalyzes its conversion to 5,10-methenyltetrahydrofolate. Because 5-FCL is mitochondrial in plants and mitochondrial SHMT is central to photorespiration, we examined the impact of an insertional mutation in the Arabidopsis 5-FCL gene (At5g13050) under photorespiratory (30 and 370 micromol of CO2 mol(-1)) and non-photorespiratory (3200 micromol of CO2 mol(-1)) conditions. The mutation had only mild visible effects at 370 micromol of CO2 mol(-1), reducing growth rate by approximately 20% and delaying flowering by 1 week. However, the mutation doubled leaf 5-CHO-THF level under all conditions and, under photorespiratory conditions, quadrupled the pool of 10-formyl-/5,10-methenyltetrahydrofolates (which could not be distinguished analytically). At 370 micromol of CO2 mol(-1), the mitochondrial 5-CHO-THF pool was 8-fold larger in the mutant and contained most of the 5-CHO-THF in the leaf. In contrast, the buildup of 10-formyl-/5,10-methenyltetrahydrofolates was extramitochondrial. In photorespiratory conditions, leaf glycine levels were up to 46-fold higher in the mutant than in the wild type. Furthermore, when leaves were supplied with 5-CHO-THF, glycine accumulated in both wild type and mutant. These data establish that 5-CHO-THF can inhibit SHMT in vivo and thereby influence glycine pool size. However, the near-normal growth of the mutant shows that even exceptionally high 5-CHO-THF levels do not much affect fluxes through SHMT or any other folate-dependent reaction, i.e. that 5-CHO-THF is well tolerated in plants.

Dietary vitamin B-6 restriction does not alter rates of homocysteine remethylation or synthesis in healthy young women and men 1–4

The effects of vitamin B-6 status on steady-state kinetics of homocysteine metabolism in humans are unclear. The objective was to determine the effects of dietary vitamin B-6 restriction on the rates of homocysteine remethylation and synthesis in healthy humans. Primed, constant infusions of [(13)C(5)]methionine, [3-(13)C]serine, and [(2)H(3)]leucine were conducted in healthy female (n=5) and male (n=4) volunteers (20-30 y) before and after 4 wk of dietary vitamin B-6 restriction (<0.5 mg vitamin B-6/d) to establish whether vitamin B-6 status affects steady-state kinetics of homocysteine metabolism in the absence of concurrent methionine intake. Effects of dietary vitamin B-6 restriction on vitamin B-6 status, plasma amino acid concentrations, and the rates of reactions of homocysteine metabolism were assessed. Dietary vitamin B-6 restriction significantly reduced plasma pyridoxal 5-phosphate (PLP) concentrations (55.1 +/- 8.3 compared with 22.6 +/- 1.3 nmol/L; P=0.004), significantly increased plasma glycine concentrations (230 +/- 14 compared with 296 +/- 15; P=0.008), and significantly reduced basal (43%; P < 0.001) and PLP-stimulated (35%; P=0.004) lymphocyte serine hydroxymethyltransferase activities in vitro. However, the in vivo fluxes of leucine, methionine, and serine; the rates of homocysteine synthesis and remethylation (total and vitamin B-6-dependent); and the concentrations of homocysteine, methionine, and serine in plasma were not significantly affected by dietary vitamin B-6 restriction. Moderate vitamin B-6 deficiency does not significantly alter the rates of homocysteine remethylation or synthesis in healthy young adults in the absence of dietary methionine intake.

Activities of Hepatic Cytosolic and Mitochondrial Forms of Serine Hydroxymethyltransferase and Hepatic Glycine Concentration Are Affected by Vitamin B-6 Intake in Rats

Serine hydroxymethyltransferase (SHMT) is a pyridoxal phosphate (PLP)–dependent enzyme that exists as cytosolic and mitochondrial isozymes that catalyze the reversible interconversion of serine and tetrahydrofolate (THF) to glycine and 5,10-methyleneTHF. SHMT is a major source of one-carbon units for cellular metabolism, but its sensitivity to various degrees of altered vitamin B-6 nutritional status has not been determined. In this study, cytosolic and mitochondrial SHMT activities were measured in liver from rats fed dietary pyridoxine (PN) ranging from adequate to deficient levels (2, 1, 0.5, 0.1, and 0 mg PN/kg diet; n = 10 per group). Both mitochondrial and cytosolic SHMT activities increased (P < 0.001) with increasing dietary PN over this range, and activities were a linear function of liver PLP concentration. Mitochondrial SHMT comprised ∼70% of total activity. Assays conducted with and without in vitro addition of PLP indicated that total SHMT (apo- and holoenzyme forms) varied with dietary PN for each isoform, but that the proportion of each present as the apoenzyme was not affected by PN intake. This aspect of SHMT nutritional regulation differs from that of many other PLP-dependent enzymes. Hepatic glycine concentration was inversely related to vitamin B-6 intake (P < 0.05), which suggests a functional effect of altered SHMT activity. Overall these results demonstrate the potential for disruption of SHMT-mediated one-carbon metabolism by inadequate vitamin B-6 intake.

Low Serine Hydroxymethyltransferase Activity in the Human Placenta Has Important Implications for Fetal Glycine Supply

Glycine is essential for fetal development, but in both sheep and human pregnancy, little is transported directly from the mother to the fetus, indicating that fetal glycine is derived from other sources. In the sheep, placental conversion of maternal serine by serine hydroxymethyltransferase (SHMT) provides almost all the glycine transported to the fetus. Although mRNA for mitochondrial and cytoplasmic SHMT has been detected in human placenta, it is not known whether substantial placental conversion of serine to glycine occurs in species other than sheep. We determined SHMT activity in human, rat, and sheep placenta by measuring conversion of [3-(14)C]serine to (14)C-methylene tetrahydrofolate. Compared with term human placenta, SHMT activity per gram of placenta was 5.1-fold higher in term rat placenta and 24.1-fold higher in term sheep placenta. In sheep placenta, SHMT activity per gram of placenta increased 2.1-fold between mid-gestation and term. In human placenta, placental SHMT activity was similar 8 wk post conception and at term. The low activity of SHMT in the human and rat placenta suggests that, unlike in the sheep, placental conversion of serine to glycine is not a major source of fetal glycine in these species.

CsgD, a regulator of curli and cellulose synthesis, also regulates serine hydroxymethyltransferase synthesis in Escherichia coli K-12.

The homologous CsgD and AgfD proteins are members of the FixJ/UhpA/LuxR family and are proposed to regulate curli (thin aggregative fibres) and cellulose production by Escherichia coli and Salmonella enterica serovar Typhimurium, respectively. A plasmid containing part of the csgD gene was isolated during a screen for multicopy suppressors of glycine auxotrophy caused by deleting the folA gene in E. coli. The sequence of the plasmid suggests it encodes a chimaeric protein. Plasmids containing the intact csgD or agfD gene also caused suppression. Cells transformed with the recombinant plasmids contained higher serine hydroxymethyltransferase (SHMT) activity than controls. The increase could also be monitored by assaying beta-galactosidase activity from a reporter strain with part of the SHMT gene, glyA, fused to lacZ. The increase in SHMT activity was sufficient to correct the glycine auxotrophy of strains lacking folA. The recombinant plasmids also enabled K-12 strains that are not curli-proficient to make curli. Curlin, the major component of curli, contains more glycine than normal E. coli proteins. It is proposed that CsgD upregulates glyA to facilitate synthesis of curli. It is suggested that recombinant plasmids produce enough CsgD or chimaeric protein to titrate out a ligand that switches CsgD into its inactive form. As a result, sufficient active CsgD is present to activate genes in its regulon. It is concluded that CsgD increases expression of the glyA gene either directly or indirectly.

Metabolic engineering of acetaldehyde production by Streptococcus thermophilus.

The process of acetaldehyde formation by the yogurt bacterium Streptococcus thermophilus is described in this paper. Attention was focused on one specific reaction for acetaldehyde formation catalyzed by serine hydroxymethyltransferase (SHMT), encoded by the glyA gene. In S. thermophilus, SHMT also possesses threonine aldolase (TA) activity, the interconversion of threonine into glycine and acetaldehyde. In this work, several wild-type S. thermophilus strains were screened for acetaldehyde production in the presence and absence of L-threonine. Supplementation of the growth medium with L-threonine led to an increase in acetaldehyde production. Furthermore, acetaldehyde formation during fermentation could be correlated to the TA activity of SHMT. To study the physiological role of SHMT, a glyA mutant was constructed by gene disruption. Inactivation of glyA resulted in a severe reduction in TA activity and complete loss of acetaldehyde formation during fermentation. Subsequently, an S. thermophilus strain was constructed in which the glyA gene was cloned under the control of a strong promoter (P(LacA)). When this strain was used for fermentation, an increase in TA activity and in acetaldehyde and folic acid production was observed. These results show that, in S. thermophilus, SHMT, displaying TA activity, constitutes the main pathway for acetaldehyde formation under our experimental conditions. These findings can be used to control and improve acetaldehyde production in fermented (dairy) products with S. thermophilus as starter culture.

Identification of glyA (Encoding Serine Hydroxymethyltransferase) and Its Use Together with the Exporter ThrE To Increase l -Threonine Accumulation by Corynebacterium glutamicum

L-threonine can be made by the amino acid-producing bacterium Corynebacterium glutamicum. However, in the course of this process, some of the L-threonine is degraded to glycine. We detected an aldole cleavage activity of L-threonine in crude extracts with an activity of 2.2 nmol min(-1) (mg of protein)(-1). In order to discover the molecular reason for this activity, we cloned glyA, encoding serine hydroxymethyltransferase (SHMT). By using affinity-tagged glyA, SHMT was isolated and its substrate specificity was determined. The aldole cleavage activity of purified SHMT with L-threonine as the substrate was 1.3 micromol min(-1) (mg of protein)(-1), which was 4% of that with L-serine as substrate. Reduction of SHMT activity in vivo was obtained by placing the essential glyA gene in the chromosome under the control of P(tac), making glyA expression isopropylthiogalactopyranoside dependent. In this way, the SHMT activity in an L-threonine producer was reduced to 8% of the initial activity, which led to a 41% reduction in glycine, while L-threonine was simultaneously increased by 49%. The intracellular availability of L-threonine to aldole cleavage was also reduced by overexpressing the L-threonine exporter thrE. In C. glutamicum DR-17, which overexpresses thrE, accumulation of 67 mM instead of 49 mM L-threonine was obtained. This shows that the potential for amino acid formation can be considerably improved by reducing its intracellular degradation and increasing its export.

Ontogeny of hepatic enzymes involved in serine- and folate-dependent one-carbon metabolism in rabbits.

Serine occupies a central position in folate-dependent, one-carbon metabolism through 5,10-methylenetetrahydrofolate (MTHF) and 5-formyltetrahydrofolate (FTHF). We characterized the ontogeny of the specific activity of key enzymes involved in serine, 5,10-MTHF, and 5-FTHF metabolism: methenyltetrahydrofolate synthetase (MTHFS), MTHF reductase (MTHFR), the glycine cleavage system (GCS), methionine synthase (MS), and serine hydroxymethyltransferase (SHMT) in rabbit liver, placenta, brain, and kidney. In liver, MTHFS activity is low in the fetus (0.36 +/- 0.07 nmol. min(-1). mg protein(-1)), peaks at 3 wk (1.48 +/- 0.50 nmol. min(-1). mg protein(-1)), and then decreases to adult levels (1.13 +/- 0.32 nmol. min(-1). mg protein(-1)). MTHFR activity is highest early in gestation (24.9 +/- 2.4 nmol. h(-1). mg protein(-1)) and declines rapidly by birth (4.7 +/- 1.3 nmol. h(-1). mg protein(-1)). MS is highest during fetal life and declines after birth. Cytosolic SHMT activity does not vary during development, but mitochondrial SHMT peaks at 23 days. GCS activity is high in the fetus and the neonate, declining after weaning. In placenta and brain, all activities are low throughout gestation. Cytosolic and mitochondrial SHMT activities are low in kidney and rise after weaning, whereas MTHFS is low throughout development. These data suggest that the liver is the primary site of activity for these enzymes. Throughout development, there are multiple potential sources for production of 5,10-MTHF, but early in gestation high MTHFR activity and low MTHFS activity could reduce 5,10-MTHF availability.

Regulation of the Balance of One-carbon Metabolism inSaccharomyces cerevisiae

One-carbon metabolism in yeast is an essential process that relies on at least one of three one-carbon donor molecules: serine, glycine, or formate. By a combination of genetics and biochemistry we have shown how cells regulate the balance of one-carbon flow between the donors by regulating cytoplasmic serine hydroxymethyltransferase activity in a side reaction occurring in the presence of excess glycine. This control governs the level of 5,10-methylene tetrahydrofolate (5,10-CH(2)-H(4)folate) in the cytoplasm, which has a direct role in signaling transcriptional control of the expression of key genes, particularly those encoding the unique components of the glycine decarboxylase complex (GCV1, GCV2, and GCV3). Based on these and other observations, we propose a model for how cells balance the need to supplement their one-carbon pools when charged folates are limiting or when glycine is in excess. We also propose that under normal conditions, cytoplasmic 5,10-CH(2)-H(4)folate is mainly directed to generating methyl groups via methionine, whereas one-carbon units generated from glycine in mitochondria are more directed to purine biosynthesis. When glycine is in excess, 5, 10-CH(2)-H(4)folate is decreased, and the regulation loop shifts the balance of generation of one-carbon units into the mitochondrion.

Vitamin B-6 Deficiency in Rats Reduces Hepatic Serine Hydroxymethyltransferase and Cystathionine β-Synthase Activities and Rates of In Vivo Protein Turnover, Homocysteine Remethylation and Transsulfuration

Vitamin B-6 deficiency causes mild elevation in plasma homocysteine, but the mechanism has not been clearly established. Serine is a substrate in one-carbon metabolism and in the transsulfuration pathway of homocysteine catabolism, and pyridoxal phosphate (PLP) plays a key role as coenzyme for serine hydroxymethyltransferase (SHMT) and enzymes of transsulfuration. In this study we used [(2)H(3)]serine as a primary tracer to examine the remethylation pathway in adequately nourished and vitamin B-6-deficient rats [7 and 0.1 mg pyridoxine (PN)/kg diet]. [(2)H(3)]Leucine and [1-(13)C]methionine were also used to examine turnover of protein and methionine pools, respectively. All tracers were injected intraperitoneally as a bolus dose, and then rats were killed (n = 4/time point) after 30, 60 and 120 min. Rats fed the low-PN diet had significantly lower growth and plasma and liver PLP concentrations, reduced liver SHMT activity, greater plasma and liver total homocysteine concentration, and reduced liver S-adenosylmethionine concentration. Hepatic and whole body protein turnover were reduced in vitamin B-6-deficient rats as evidenced by greater isotopic enrichment of [(2)H(3)]leucine. Hepatic [(2)H(2)]methionine production from [(2)H(3)]serine via cytosolic SHMT and the remethylation pathway was reduced by 80.6% in vitamin B-6 deficiency. The deficiency did not significantly reduce hepatic cystathionine-beta-synthase activity, and in vivo hepatic transsulfuration flux shown by production of [(2)H(3)]cysteine from the [(2)H(3)]serine increased over twofold. In contrast, plasma appearance of [(2)H(3)]cysteine was decreased by 89% in vitamin B-6 deficiency. The rate of hepatic homocysteine production shown by the ratio of [1-(13)C]homocysteine/[1-(13)C]methionine areas under enrichment vs. time curves was not affected by vitamin B-6 deficiency. Overall, these results indicate that vitamin B-6 deficiency substantially affects one-carbon metabolism by impairing both methyl group production for homocysteine remethylation and flux through whole-body transsulfuration.

Rapid uptake and degradation of glycine by astroglial cells in culture: synthesis and release of serine and lactate.

Free glycine is known to have vital functions in the mammalian brain, where it serves mainly as both neurotransmitter and neuromodulator. Despite its importance, little is known about the metabolic pathways of glycine synthesis and degradation in the central nervous system. In this study, the pathway of glycine metabolism in astroglia-rich primary cultures from rat brain was examined. The cells were allowed to degrade glycine in the presence of [U-(14)C]glycine, [U-(13)C]glycine or [(15)N]glycine. The resulting intra- and extracellular metabolites were analyzed both by high-performance liquid chromatography and by (13)C/(15)N nuclear magnetic resonance spectroscopy. Glycine was rapidly consumed in a process obeying first-order kinetics. The initial glycine consumption rate was 0.47 nmol per mg protein. The half-life of glycine radiolabel in the incubation medium was shorter than that of glycine mass. This suggests that glycine is produced from endogenous sources and released simultaneously with glycine uptake and metabolism. As the main metabolites of the glycine carbon skeleton in astroglia-rich primary cultures from rat brain, serine and lactate were released during glycine consumption. The main metabolite containing the glycine amino nitrogen was glutamine. To establish a metabolic pathway from glycine to serine in neural tissue, homogenates of rat brain and of neural primary cultures were assayed for their content of serine hydroxymethyltransferase (SHMT) and glycine cleavage system (GCS). SHMT activity was present in homogenates of rat brain as well as of astroglia-rich and neuron-rich primary cultures, whereas GCS activity was detectable only in homogenates of rat brain and astroglia-rich primary culture. Of the two known SHMT isoenzymes, only the mitochondrial form was found in rat brain homogenate. It is proposed that, in neural tissue, glycine is metabolized by the combined action of SHMT and the GCS. Owing to the absence of the GCS from neurons, astrocytes appear to be the only site of this part of glycine metabolism in brain. However, neurons are able to utilize as energy source the lactate formed by astroglial cells in this metabolic pathway.

Purification, crystallization and preliminary X-ray analysis of human recombinant cytosolic serine hydroxymethyltransferase.

As an enzyme of the thymidylate synthase cycle, serine hydroxymethyltransferase (SHMT) has a key role in nucleotide biosynthesis. Elevated activities of SHMT have been correlated with the increased demand for nucleotide biosynthesis in tumors of human and rodent origin, making this enzyme a novel target for cancer chemotherapy. Here the purification and crystallization of recombinant human cytosolic SHMT are reported. Crystals belong to space group P6222 or P6422 with cell parameters a = b = 155.0, c = 235.5 A and diffract to at least 3.0 A resolution.

The crystal structure of human cytosolic serine hydroxymethyltransferase: a target for cancer chemotherapy.

Serine hydroxymethyltransferase (SHMT) is a ubiquitous enzyme found in all prokaryotes and eukaryotes. As an enzyme of the thymidylate synthase metabolic cycle, SHMT catalyses the retro-aldol cleavage of serine to glycine, with the resulting hydroxymethyl group being transferred to tetrahydrofolate to form 5, 10-methylene-tetrahydrofolate. The latter is the major source of one-carbon units in metabolism. Elevated SHMT activity has been shown to be coupled to the increased demand for DNA synthesis in rapidly proliferating cells, particularly tumour cells. Consequently, the central role of SHMT in nucleotide biosynthesis makes it an attractive target for cancer chemotherapy. We have solved the crystal structure of human cytosolic SHMT by multiple isomorphous replacement to 2.65 A resolution. The monomer has a fold typical for alpha class pyridoxal 5'-phosphate (PLP) dependent enzymes. The tetramer association is best described as a 'dimer of dimers' where residues from both subunits of one 'tight' dimer contribute to the active site. The crystal structure shows the evolutionary relationship between SHMT and other alpha class PLP-dependent enzymes, as the fold is highly conserved. Many of the results of site-directed mutagenesis studies can easily be rationalised or re-interpreted in light of the structure presented here. For example, His 151 is not the catalytic base, contrary to the findings of others. A mechanism for the cleavage of serine to glycine and formaldehyde is proposed.

Investigation of serine hydroxymethyltransferase in methanogens

The cofactor specificity of serine hydroxymethyltransferase (SHMT) activities was tested in extracts of several methanogens using tetrahydromethanopterin (H4MPt) from Methanobacterium thermoautotrophicum Marburg, tetrahydrosarcinapterin (H4SPt) from Methanosarcina barkeri, and tetrahydrofolate (H4folate) as the potential C1 carrier. In Methanosphaera stadtmanae and Methanococcus thermolithotrophicus, the activities were H4MPt dependent. In Methanospirillum hungatei GP1, Methanosaeta concilii, Methanolobus tindarius, and Methanosarcina barkeri Fusaro, the activities were strictly H4folate dependent. H4SPt was reactive with the SHMT of Methanosphaera stadtmanae but not with that of Methanosarcina barkeri. In both Methanosarcina barkeri and Methanospirillum hungatei, pyridoxal phosphate stimulated SHMT activity. The apparent Km values for H4folate and L-serine were 0.086 and 0.29 mM in Methanosarcina barkeri and 0.065 and 0.31 mM in Methanospirillum hungatei, respectively. Key words: tetrahydromethanopterin, tetrahydrofolate, Archaea, serine hydroxymethyltransferase, methanogen.

Investigation of serine hydroxymethyltransferase in methanogens.

The cofactor specificity of serine hydroxymethyltransferase (SHMT) activities was tested in extracts of several methanogens using tetrahydromethanopterin (H4MPt) from Methanobacterium thermoautotrophicum Marburg, tetrahydrosarcinapterin (H4SPt) from Methanosarcina barkeri, and tetrahydrofolate (H4folate) as the potential C1 carrier. In Methanosphaera stadtmanae and Methanococcus thermolithotrophicus, the activities were H4MPt dependent. In Methanospirillum hungatei GP1, Methanosaeta concilii, Methanolobus tindarius, and Methanosarcina barkeri Fusaro, the activities were strictly H4folate dependent. H4SPt was reactive with the SHMT of Methanosphaera stadtmanae but not with that of Methanosarcina barkeri. In both Methanosarcina barkeri and Methanospirillum hungatei, pyridoxal phosphate stimulated SHMT activity. The apparent K(m) values for H4folate and L-serine were 0.086 and 0.29 mM in Methanosarcina barkeri and 0.065 and 0.31 mM in Methanospirillum hungatei, respectively.

Decrease in glycolate pathway enzyme activities in plastids and peroxisomes of the albostrians mutant of barley ( Hordeum vulgare L.)

The in-vitro activities of various enzymes involved in the glycolate pathway were analyzed in crude leaf extracts of the albostrians mutant of barley. This mutant line is characterized by the lack of plastid ribosomes in white leaf tissue, thus causing a complex modification of nuclear gene expression. In white leaf cells the activities of the plastidic enzymes phosphoglycolate phosphatase and glycerate kinase, as well as the activities of the peroxisomal enzymes glycolate oxidase, catalase, and hydroxypyruvate reductase, are drastically reduced by as much as 80–99% compared to green wild-type leaf tissue. The activity of plastidic NADPH-glyoxylate reductase, the total activity of serine hydroxymethyltransferase and the activity of mitochondrial fumarase was reduced by only 30–52%. The activities of the glyoxysomal enzymes isocitrate lyase and malate synthase were neither detectable in green nor white mutant leaf tissue. Electron microscopy showed fewer and smaller peroxisomes in white leaf tissue, accounting for at least part of the reduction of peroxisomal enzyme activities. Northern blot analysis for the catalase and glycolate oxidase transcripts revealed strongly reduced levels of steady-state mRNA accumulation. It is suggested that a putative plastidic signal(s) influences the expression of nuclear-encoded plastidic and peroxisomal enzymes of the glycolate pathway.

5-Formyltetrahydrofolate Regulates Homocysteine Remethylation in Human Neuroblastoma

The metabolic role of 5-formyltetrahydrofolate is not known; however, it is an inhibitor of several folate-dependent enzymes including serine hydroxymethyltransferase. Methenyltetrahydrofolate synthetase (MTHFS) is the only enzyme known to metabolize 5-formyltetrahydrofolate and catalyzes the conversion of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate. In order to address the function of 5-formyltetrahydrofolate in mammalian cells, intracellular 5-formyltetrahydrofolate levels were depleted in human 5Y neuroblastoma by overexpressing the human cDNA encoding MTHFS (5YMTHFS cells). When cultured with 2 mM exogenous glycine, the intracellular serine and glycine concentrations in 5YMTHFS cells are elevated approximately 3-fold relative to 5Y cells; 5YMTHFS cells do not contain measurable levels of free methionine and display a 30-40% decrease in cell proliferation rates compared with 5Y cells. Medium supplemented with pharmacological levels of exogenous folinate or methionine ameliorated the glycine induced growth inhibition. Analysis of the folate derivatives demonstrated that 5-methyltetrahydrofolate accounts for 30% of total cellular folate in 5Y cells when cultured with 5 mM exogenous glycine. 5YMTHFS cells do not contain detectable levels of 5-methyltetrahydrofolate under the same culture conditions. These results suggest that 5-formyltetrahydrofolate inhibits serine hydroxymethyltransferase activity in vivo and that serine synthesis and homocysteine remethylation compete for one-carbon units in the cytoplasm.