Selection of SALMONELLA TYPHIMURIUM Mutants with Altered Serine Transhydroxymethylase Regulation

In Salmonella typhimurium the glyA gene product, serine transhydroxymethylase (E.C. 2.1.2.1.; L-serine:tetrahydrofolate-5,10-hydroxymethyltransferase) is responsible for the interconversion of serine and glycine. This reaction also provides the cell with one-carbon units from the 5,10-methylene-tetrahydrofolate formed during glycine synthesis. Despite the importance of this enzyme, however, no mutants in which its regulation has been specificially altered have been isolated. To isolate such mutants, we have devised a selection procedure using a strain (glyA951) in which the serine transhydroxymethylase activity is reduced. When this enzyme is completely repressed, the mutant requires gylcine for growth. Revertants which retain the glyA951 lesion, but no longer require glycine, have been isolated and the serine transhydroxymethylase regulation examined. One revertant has a 7-fold elevated serine transhydroxymethylase level, which can be repressed the normal amount (about 5-fold) when the cells are grown in supplemented media. Another revertant has only a 2-fold higher serine transhydroxymethylase level; however, the amount of repression is reduced. The new lesions in both mutants cotransduce with the glyA gene and are distinct from other mutations that alter the regulation of both serine transhydroxymethylase and the methionine biosyntheitc enzymes.     

Serine transhydroxymethylase in methionine biosynthesis in Saccharomyces cerevisiae

Serine transhydroxymethylase appears to be the first enzyme in the synthesis of the methyl group of methionine. Properties of serine transhydroxymethylase activity as assayed by the production of formaldehyde were correlated with properties of cell-free extracts for the methylation of homocysteine deriving the methyl group from the beta-carbon of serine. The reaction required pyridoxal phosphate and tetrahydrofolic acid, and was characterized in cell-free extracts with respect to Michaelis constant, pH optimum, incubation time, and optimal enzyme concentration. The activity was sensitive to inhibition by methionine, and to a much greater extent by S-adenosylmethionine. Serine transhydroxymethylase and the methylation of homocysteine reactions were not repressed by methionine and were stimulated by glycine. The activities of cell-free extracts for these reactions were significantly higher in cells in exponential than in stationary growth. When cells were grown in 10 mm glycine, the activities remained high throughout the culture cycle. The data indicated that glycine rather than methionine is involved in the control of the formation of the enzyme.     

Influence of methionine biosynthesis on serine transhydroxymethylase regulation in Salmonella typhimurium LT2.

The enzyme serine transhydroxymethylase (EC 2.1.2.1; L-serine:tetrahydrofolate-5,10-hydroxymethyltransferase) is responsible both for the synthesis of glycine from serine and production of the 5,10-methylenetetrahydrofolate necessary as a methyl donor for methionine synthesis. Two mutants selected for alteration in serine transhydroxymethylase regulation also have phenotypes characteristic of metK (methionine regulatory) mutants, including ethionine, norleucine, and alpha-methylmethionine resistance and reduced levels of S-adenosylmethionine synthetase (EC 2.5.1.6; adenosine 5'-triphosphate:L-methionine S-adenosyltransferase) activity. Because this suggested the existence of a common regulatory component, the regulation of serine transhydroxymethylase was examined in other methionine regulatory mutants (metK and metJ mutants). Normally, serine transhydroxymethylase levels are repressed three- to sixfold in cells grown in the presence of serine, glycine, methionine, adenine, guanine, and thymine. This does not occur in metK and metJ mutants; thus, these mutations do affect the regulation of both serine transhydroxymethylase and the methionine biosynthetic enzymes. Lesions in the metK gene have been reported to reduce S-adenosylmethionine synthetase levels. To determine whether the metK gene actually encodes for S-adenosylmethionine synthetase, a mutant was characterized in which this enzyme has a 26-fold increased apparent Km for methionine. This mutation causes a phenotype associated with metK mutants and is cotransducible with the serA locus at the same frequency as metK lesions. Thus, the affect of metK mutations on the regulation of glycine and methionine synthesis in Salmonella typhimurium appears to be due to either an altered S-adenosylmethionine synthetase or altered S-adenosylmethionine pools.     

Serine Transhydroxymethylase Activity in Vertebrate Retina

Abstract: The presence of serine transhydroxymethylase (STHM; EC 2.1.2.1) in crude homogenates of rat, guinea pig and goat retina has been demonstrated. There was a variation in the levels of retinal STHM activity in the three species. Substantial STHM activity was found in all the three retinal preparations in the absence of pyridoxal-5-phosphate in the incubation medium. There was direct correlation ( r = 0.995; p < 0.001) between the STHM activity and the glycine content in rat telencephalon, midbrain, medulla-pons, spinal cord and retina. These findings suggest that STHM is probably an important enzyme for the synthesis of neural glycine in the vertebrate retina.     

The metabolism of serine and glycine in mutant lines of Chinese hamster ovary cells

This report describes studies of mutant lines of cultured Chinese hamster ovary cells that have different levels of serine transhydroxymethylase (EC 2.1.2.1). This enzyme, which splits serine to yield glycine and N⁵,N¹⁰-methylene tetrahydrofolic acid, is found in both the mitochondria and cytosol of these cells (see Chasin et al. (1974) Proc. Nat. Acad. Sci. USA71, 718–722). Our experiments with these mutant lines have established a correlation among the amount of mitochondrial serine transhydroxymethylase, the intracellular glycine concentration, and the extent that exogenous serine increases the glycine pool. Limited amino acid incorporation into protein occurred with all cell lines, but in contrast to the glycine-requiring mutant line 51-11, revertants that no longer required glycine for growth showed increased incorporation when the medium was supplemented with serine. These results indicate that normally the mitochondrial serine transhydroxymethylase together with the intracellular serine concentration regulate the supply of glycine and under certain conditions can control the rate of protein synthesis. Additional experiments with radioactive serine and glycine have shown that the mitochondrial serine transhydroxymethylase regulates the interconversion of these amino acids as well as serine oxidation. Calculations based on the ¹⁴CO₂ produced from l-[¹⁴C]serine by the mutant and parental cell lines indicate that approximately 50% of the serine oxidized is initially converted to glycine and an oxidizable one-carbon unit.     

Mechanistic, inhibitory and stereochemical studies on cytoplasmic and mitochondrial serine transhydorxymethylases.

By using cytoplasmic and mitochondrial serine transhydroxymethylase isoenzymes from rabbit liver, it was shown that both enzymes exhibited similar ratios of serine transhydroxymethylase/threonine aldolase activities. Both enzymes catalysed the removal of the pro-S hydrogen atom of glycine, which was greatly enhanced by the presence of tetrahydrofolate. The cytoplasmic as well as the mitochondrial enzyme catalysed the synthesis of serine from glycine and [3H2]formaldehyde in the absence of tetrahydrofolate. The results are consistent with our previous suggestion that a role of tetrahydrofolate in the serine transhydroxymethylase reaction is to transport formaldehyde in and out of the active site (Jordan & Akhtar, 1970). The isoenzymes, however, showed remarkable differences in their inactivation by inhibitors. The serine transhydroxymethylase as well as the threonine aldolase activities of the cytoplasmic enzyme were inactivated in a similar fashion by chloroacetaldehyde, iodoacetamide, bromopyruvate and glycidaldehyde (2,3-epoxypropionaldehyde). These inhibitors had no effect on the two activities of the mitochondrial enzyme. The rate of inactivation of the cytoplasmic enzyme by glycidaldehyde was enhanced by the presence of glycine but decreased by the presence of serine. The implications of these results to the mechanism of catalysis and the nature of the active site of the enzymes are discussed.     

Regulation of One-Carbon Biosynthesis and Utilization in Escherichia coli

The regulation of several enzymes involved in one-carbon metabolism was studied in a mutant of Escherichia coli K-12 defective in S-adenosylmethionine synthetase. The mutant that was reported to have a low endogenous concentration of S-adenosylmethionine had elevated levels of N-5, 10-methylene tetrahydrofolate reductase and serine transhydroxymethylase, but the level of N-5, 10-methylene tetrahydrofolate dehydrogenase was not altered. These results suggest that S-adenosylmethionine plays a role in the regulation of one-carbon production and utilization. An enzyme system that cleaved glycine to one-carbon units was demonstrated. The enzymes responsible for the cleavage of glycine were induced by exogenous glycine but were independent of S-adenosylmethionine or purine levels in the cell.     

The mechanism of action of serine transhydroxymethylase

1. The preparation of stereospecifically tritiated glycines and the determination of their absolute configurations by the use of d-amino acid oxidase are described. 2. The reaction catalysed by serine transhydroxymethylase, which results in the conversion of glycine into serine, has been separated into at least four partial reactions. It is suggested that the first event in this conversion is the formation of a Schiff base intermediate of glycine and pyridoxal phosphate. The next important step involves the removal of the 2S-hydrogen atom of glycine to give a carbanion intermediate. Experiments pertinent to the mechanism of conversion of this carbanion intermediate into serine are described. 3. The enzyme preparation catalysing the conversion of glycine into serine also participates in the conversion of glycine into threonine and allothreonine. In both these conversions, glycine → serine and glycine → threonine, the 2S-hydrogen atom of glycine is eliminated and the 2R-hydrogen atom of glycine is retained. 4. In the light of these experiments the mechanism of action of serine transhydroxymethylase is discussed. It is suggested that methylenetetrahydrofolate is the carrier of formaldehyde, from which formaldehyde may be liberated at the active site of the enzyme, thus allowing the overall reaction to take place.     

Photorespiration-deficient Mutants of Arabidopsis thaliana Lacking Mitochondrial Serine Transhydroxymethylase Activity

Three allelic mutants of Arabidopsis thaliana which lack mitochondrial serine transhydroxymethylase activity due to a recessive nuclear mutation have been characterized. The mutants were shown to be deficient both in glycine decarboxylation and in the conversion of glycine to serine. Glycine accumulated as an end product of photosynthesis in the mutants, largely at the expense of serine, starch, and sucrose formation. The mutants photorespired CO₂ at low rates in the light, but this evolution of photorespiratory CO₂ was abolished by provision of exogenous NH₃. Exogenous NH₃ was required by the mutants for continued synthesis of glycine under photorespiratory conditions. These and related results with wild-type Arabidopsis suggested that glycine decarboxylation is the sole site of photorespiratory CO₂ release in wild-type plants but that depletion of the amino donors required for glyoxylate amination may lead to CO₂ release from direct decarboxylation of glyoxylate. Photosynthetic CO₂ fixation was inhibited in the mutants under atmospheric conditions which promote photorespiration but could be partially restored by exogenous NH₃. The magnitude of the NH₃ stimulation of photosynthesis indicated that the increase was due to the suppression of glyoxylate decarboxylation. The normal growth of the mutants under nonphotorespiratory atmospheric conditions indicates that mitochondrial serine transhydroxymethylase is not required in C₃ plants for any function unrelated to photorespiration.     

Serine transhydroxymethylase isoenzymes from a facultative methylotroph.

Two serine transhydroxymethylase activities have been purified from a facultative methylotrophic bacterium. One enzyme predominates when the organism is grown on methane or methanol as the sole carbon and energy source, whereas the second enzyme is the major isoenzyme found when succinate is used as the sole carbon and energy source. The enzyme from methanol-grown cells is activated by glyoxylate, is not stimulated by Mg2+, Mn2+, or Zn2+, and has four subunits of 50,000 molecular weight each. The enzyme from succinate-grown cells is not activated by glyoxylate and is stimulated by Mg2+, Mn2+, and Zn2+, and sodium dodecyl sulfate-acrylamide gel electrophoresis indicates that this enzyme has subunit molecular weight of 100,000, the same as the molecular weight obtained for the active enzyme. Cells grown in the presence of both methanol and succinate incorporate less methanol carbon per unit time than cells grown on methanol and have a lower specific activity of the glyoxylate-activated enzyme than methanol-grown cells. Adenine, glyoxylate, or trimethoprim in the growth medium causes an increased level of serine transhydroxymethylase in both methanol- and succinate-grown cells by stimulating the synthesis of the glyoxylate-activated enzyme.     

Genetics of the Synthesis of Serine from Glycine and the Utilization of Glycine as Sole Nitrogen Source by Saccharomyces Cerevisiae

Saccharomyces cerevisiae can grow on glycine as sole nitrogen source and can convert glycine to serine via the reaction catalyzed by the glycine decarboxylase multienzyme complex (GDC). Yeast strains with mutations in the single gene for lipoamide dehydrogenase (lpd1) lack GDC activity, as well as the other three 2-oxoacid dehydrogenases dependent on this enzyme. The LPD1 gene product is also required for cells to utilize glycine as sole nitrogen source. The effect of mutations in LPD1 (L-subunit of GDC), SER1 (synthesis of serine from 3-phosphoglycerate), ADE3 (cytoplasmic synthesis of one-carbon units for the serine synthesis from glycine), and all combinations of each has been determined. The results were used to devise methods for isolating mutants affected either in the generation of one-carbon units from glycine (via GDC) or subsequent steps in serine biosynthesis. The mutants fell into six complementation groups (gsd1-6 for defects in conversion of glycine to serine). Representatives from three complementation groups were also unable to grow on glycine as sole nitrogen source (gsd1-3). Assays of the rate of glycine uptake and decarboxylation have provided insights into the nature of the mutations.     

Derivation of glycine from threonine in Escherichia coli K-12 mutants.

Escherichia coli AT2046 has been shown previously to lack the enzyme serine transhydroxymethylase and to require exogenous glycine for growth as a consequence. Strains JEV73 and JEV73R, mutants derived from strain AT2046, are shown here to be serine transhydroxymethylase deficient, but able to derive their glycine from endogenously synthesized threonine. Leucine is shown to be closely involved in the regulation of biosynthesis of glycine, to spare glycine in strain AT2046T, to replace glycine in strain JEV73, and to increase threonine conversion to glycine in a representative prototroph of E. coli. An interpretation of strains JEV73 and JEV73R as regulatory mutants of strain AT2046 is given. A hypothesis as to the role of leucine as a signal for nitrogen scavenging is suggested.     

Serine biosynthesis and its regulation in Bacillus subtilis

Cell-free extracts of Bacillus subtilis strains GSY and 168 convert (14)C-phosphoglycerate to (14)C-serine phosphate and (14)C-serine. These reactions indicate a functional phosphorylated pathway for serine biosynthesis in these cells. The addition of serine to the incubation mixture inhibited the formation of both radioactive products. Extracts of mutant strains that require serine for growth lacked the capacity to synthesize serine phosphate, confirming that the phosphorylated pathway was the only functional pathway available for serine synthesis. Serine phosphate phosphatase and phosphoglycerate dehydrogenase activity were demonstrated in cell extracts, and the phosphoglycerate dehydrogenase was shown to be inhibited specifically by l-serine. The extent of serine inhibition increased when the temperature was raised from 25 to 37 C, and the thermal stability of the enzyme was enhanced by the presence of the inhibitor serine or the coenzyme reduced nicotinamide adenine dinucleotide. At 37 C the curve representing the relationship between phosphoglycerate concentration and enzyme velocity was biphasic, and the serine inhibition which was competitive at low substrate concentrations became noncompetitive at higher concentrations.     

Serine transhydroxymethylase. Equilibrium binding of folate analogs as active site probes

Formation of a quinoid-like structure within the glycyl-pyridoxal phosphate moiety of serine transhydroxymethylase (5,10-methylenetetrahydrofolate: glycine hydroxymethyltransferase, EC 2.1.2.1) is dependent upon the dissociation of the 2-S hydrogen of glycine which in turn requires the presence of tetrahydrofolate or analogs thereof. Equilibrium binding studies with the series folate, dihydrofolate, and tetrahydrofolate showed that reduction of the pteridine ring enhances both quinoid formation and binding. A 5,8-deazafolate series showed that modifications in the 4 position, 10 position and the glutamyl position yield interrelated alterations of quinoid formation which could not be correlated with binding.     

Effect of Oxygen and Carbon Dioxide on Photorespiratory Flux Determined from Glycine Accumulation in a Mutant of Arabidopsis thaliana

Exposure to atmospheric conditions which promote photorespirationstrongly inhibits photosynthesis in a mutant of Arabidopsislacking mitochondrial serine transhydroxymethylase activity,and glycine accumulates as a stable end-product of photorespiratorycarbon and nitrogen flow. By providing exogenous serine andammonia to leaves of the mutant, wild-type photosynthesis ratescan be temporarily maintained in the absence of photorespiratoryCO₂ evolution. In these circumstances, the rate of glycine accumulationprovides a direct measure of photorespiratory flux which isnot complicated by the efflux and refixation of photorespiredCO₂, the dilution of radioactive label by endogenous metabolicpools, or non-specific effects of metabolic inhibitors. At thestandard atmospheric concentration of CO₂, the rate of glycineaccumulation in the mutant was proportional to the oxygen concentration,amounting to 53% of the rate of gross CO₂-fixation at 21% O₂.At normal levels of O₂, glycine accumulation was maximal atabout 475 µl CO₂1^–1 and was reduced at higher orlower CO₂ concentrations, being almost abolished at 3000µ1CO₂1^–1. These observations are discussed in the contextof a model of photorespiration based on the properties of ribulose1, 5-bisphosphate carboxylase/oxygenase, and in relation tothe results of previous attempts to measure photorespiration.Preliminary evidence from ¹⁴CO₂-labelling experiments whichsuggests a non-photorespiratory pathway of serine synthesisis also presented. Key words: Arabidopsis mutant, Photorespiration, Serine transhydroxymethylase     

Serine hydroxymethyltransferase catalyzes the hydrolysis of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate

The combined activities of rabbit liver cytosolic serine hydroxymethyltransferase and C1-tetrahydrofolate synthase convert tetrahydrofolate and formate to 5-formyltetrahydrofolate. In this reaction C1-tetrahydrofolate synthase converts tetrahydrofolate and formate to 5,10-methenyltetrahydrofolate, which is hydrolyzed to 5-formyltetrahydrofolate by a serine hydroxymethyltransferase-glycine complex. Serine hydroxymethyltransferase, in the presence of glycine, catalyzes the conversion of chemically synthesized 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate with biphasic kinetics. There is a rapid burst of product that has a half-life of formation of 0.4 s followed by a slower phase with a completion time of about 1 h. The substrate for the burst phase of the reaction was shown not to be 5,10-methenyltetrahydrofolate but rather a one-carbon derivative of tetrahydrofolate which exists in the presence of 5,10-methenyltetrahydrofolate. This derivative is stable at pH 7 and is not an intermediate in the hydrolysis of 5,10-methenyltetrahydrofolate to 10-formyltetrahydrofolate by C1-tetrahydrofolate synthase. Cytosolic serine hydroxymethyltransferase catalyzes the hydrolysis of 5,10-methenyltetrahydrofolate pentaglutamate to 5-formyltetrahydrofolate pentaglutamate 15-fold faster than the hydrolysis of the monoglutamate derivative. The pentaglutamate derivative of 5-formyltetrahydrofolate binds tightly to serine hydroxymethyltransferase and dissociates slowly with a half-life of 16 s. Both rabbit liver mitochondrial and Escherichia coli serine hydroxymethyltransferase catalyze the conversion of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate at rates similar to those observed for the cytosolic enzyme. Evidence that this reaction accounts for the in vivo presence of 5-formyltetrahydrofolate is suggested by the observation that mutant strains of E. coli, which lack serine hydroxymethyltransferase activity, do not contain 5-formyltetrahydrofolate, but both these cells, containing an overproducing plasmid of serine hydroxymethyltransferase, and wild-type cells do have measurable amounts of this form of the coenzyme.     

Serine transhydroxymethylase: mechanism of aldolase activation by folate.

Affinity chromatographic methods have been developed to purify beef liver serine transhydroxymethylase. This enzyme catalyzes both the transfer of aldehydic groups from β-hydroxy amino acids to tetrahydrofolate and the cleavage of β-hydroxy amino acids yielding the free aldehyde. Tetrahydrofolate, folate, and a quinazoline analog of isofolate were found to be activators of the β-phenylserine aldolase reaction catalyzed by serine transhydroxymethylase. Activation by folate was maximal at 20 μm and higher concentrations diminished the activation. Evidence is presented suggesting that folate does not activate by providing an acceptor site for the aldehydic groups. Equilibrium binding studies showed that folate and tetrahydrofolate can bind the enzyme with essentially the same affinity. Double-reciprocal plots with β-phenylserine from steady-state kinetic experiments did not yield a $\text{1}\text{v}{}_1$, intercept effect except at high folate concentrations. A mechanism is proposed in which folate binds readily to the enzymic active site, facilitating β-phenylserine binding. Folate is subsequently lost, at least partially, prior to product release and complete enzymic turnover.     

The biosynthesis of serine and glycine in Pseudomonas AM1 with special reference to growth on carbon sources other than C1 compounds

1. A mutant, 20S, of Pseudomonas AM1 was obtained that requires a supplement of serine to grow on succinate, lactate or ethanol. This mutant lacks phosphoserine phosphatase and revertants to wild-type phenotype regained this enzymic activity showing that the phosphorylated pathway of serine biosynthesis is necessary for growth on these three substrates. 2. The requirement for supplemental serine by mutant 20S could be met by glycine, suggesting that Pseudomonas AM1 can obtain C(1) units from glycine. 3. Mutant 20S grows on C(1) compounds at a lower rate compared with the wild type. Supplementation with serine stimulated the growth rate of the mutant suggesting that the phosphorylated pathway of serine biosynthesis plays some role, but not an essential role, during growth on C(1) compounds. 4. A mutant, 82G, was obtained that requires a supplement of glycine to grow on succinate, lactate or ethanol. When grown in such supplemented media, the mutant lacks serine hydroxymethyltransferase and revertants to wild-type phenotype regained enzymic activity showing that during growth on succinate, lactate or ethanol, glycine is made from serine via serine hydroxymethyltransferase, and that the organism can obtain C(1) units from glycine. 5. Mutant 82G grew on methanol and then contained serine hydroxymethyltransferase suggesting that this enzyme is necessary for growth on C(1) compounds and that Pseudomonas AM1 may synthesize two such enzymes, one used in growth on C(1) compounds, the other used in growth on other substrates. Mutant 82G might lack the latter enzyme. 6. Phosphoglycerate dehydrogenase is specifically inhibited by l-serine and the regulatory implications of this are discussed.     

Serine transhydroxymethylase in developing mouse brain

Abstract— Serine transhydroxymethylase in extracts from mouse brain declines in specific activity during the first 2 weeks of postnatal life. This decrease in potential for synthesis of methylene tetrahydrofolate from serine is most probably compatible with the declining postnatal rates of protein and nucleic acid synthesis.     

Threonine as a carbon source for Escherichia coli.

Threonine can be used aerobically as the sole source of carbon and energy by mutants of Escherichia coli K-12. The pathway used involves the conversion of threonine via threonine dehydrogenase to aminoketobutyric acid, which is further metabolized by aminoketobutyric acid ligase, forming acetyl coenzyme A and glycine. A strain devoid of serine transhydroxymethylase uses this pathway and excretes glycine as a waste product. Aminoketobutyric acid ligase activity was demonstrated after passage of crude extracts through Sephadex G100.