Escherichia coli K12 mutants defective in the glycine cleavage enzyme system

Two routes of one-carbon biosynthesis have been described in Escherichia coli K12. One is from serine via the serine hydroxymethyltransferase (SHMT) reaction, and the other is from glycine via the glycine cleavage (GCV) enzyme system. To isolate mutants deficient in the GCV pathway, we used a selection procedure that is based on the assumption that loss of this enzyme system in strains blocked in serine biosynthesis results in their inability to use glycine as a serine source. Mutants were accordingly isolated that grow with a serine supplement, but not with a glycine supplement. Enzyme assays demonstrated that three independently isolated mutants have no detectable GCV enzyme activity. The absence of a functional GCV pathway results in the excretion of glycine, but has no affect on the cell's primary source of one-carbon units, the SHMT reaction. The new mutations, designated gcv, were mapped between the serA and lysA genes on the E. coli chromosome.     

Cytoplasmic serine hydroxymethyltransferase regulates the metabolic partitioning of methylenetetrahydrofolate but is not essential in mice

The hydroxymethyl group of serine is a primary source of tetrahydrofolate (THF)-activated one-carbon units that are required for the synthesis of purines and thymidylate and for S-adenosylmethionine (AdoMet)-dependent methylation reactions. Serine hydroxymethyltransferase (SHMT) catalyzes the reversible and THF-dependent conversion of serine to glycine and 5,10-methylene-THF. SHMT is present in eukaryotic cells as mitochondrial SHMT and cytoplasmic (cSHMT) isozymes that are encoded by distinct genes. In this study, the essentiality of cSHMT-derived THF-activated one-carbons was investigated by gene disruption in the mouse germ line. Mice lacking cSHMT are viable and fertile, demonstrating that cSHMT is not an essential source of THF-activated one-carbon units. cSHMT-deficient mice exhibit altered hepatic AdoMet levels and uracil content in DNA, validating previous in vitro studies that indicated this enzyme regulates the partitioning of methylenetetrahydrofolate between the thymidylate and homocysteine remethylation pathways. This study suggests that mitochondrial SHMT-derived one-carbon units are essential for folate-mediated one-carbon metabolism in the cytoplasm.     

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.     

Biosynthesis of 5-hydroxy-L-tryptophan by a genetically modified strain ofEscherichia coli in presence of 5-hydroxyindole

Summary A derivative ofEscherichia coli strain W3110 with increased tryptophan synthas (TS) activity was studied in the biosynthesis of L-tryptophan and 5-hydroxy-L-tryptophan, respectively, in presence of precursors. Indole or 5-hydroxyindole was added to growing cells in minimal medium supplemented with tetracycline. The specific activity of TS for 5-hydroxyindole was about 5-fold lower compared with indole. However, this difference in enzyme activity was not observed when the specific productivity (q_p) of L-tryptophan or 5-hydroxy-L-tryptophan, which was 0.14–0.15 g (g dry wt cells)^–1 · h^–1 was determined. In minimal medium L-serine was shown to limit the production of both tryptophan and its hydroxylated derivative. In presence of L-serine, q_p, for L-tryptophan and 5-hydroxy-L-tryptophan were increased by a factor of about 3 and 2, respectively.     

Effect of vitamin B6 availability on serine hydroxymethyltransferase in MCF-7 cells

Folate-activated one-carbon units are derived from serine through the activity of the pyridoxal-phosphate (PLP)-dependent isozymes of serine hydroxymethyltransferase (SHMT). The effect of vitamin B(6) availability on the activity and expression of the human mitochondrial and cytoplasmic SHMT isozymes was investigated in human MCF-7 cells. Cells were cultured for 6 months in vitamin B(6) replete (4.9 microM pyridoxine) minimal essential medium (alphaMEM) or vitamin B(6)-deficient medium containing 49, 4.9 or 0.49 nM pyridoxine. Total cellular PLP levels and SHMT activity were reduced 72% and 7%, respectively, when medium pyridoxine was decreased from 4.9 microM to 49 nM. Cells cultured in medium containing 4.9 nM pyridoxine exhibited 75%, 27% and 60% reduced levels of PLP, SHMT activity and S-adenosylmethionine, respectively, compared to cells cultured in alphaMEM. Cytoplasmic SHMT activity and protein levels, but not mRNA levels, were decreased in cells cultured in vitamin B(6) deficient medium, whereas mitochondrial SHMT activity and protein levels were less sensitive to vitamin B(6) availability. PLP bound to cytoplasmic SHMT with a K(d)=850 nM, a value two orders of magnitude lower than previously reported for the bovine cytoplasmic SHMT isozyme. Collectively, these data indicate that vitamin B(6) restriction decreases the activity and stability of SHMT, and that the cytoplasmic isozyme is more sensitive to vitamin B(6) deficiency than the mitochondrial isozyme in MCF-7 cells.     

Modulation of the heat shock response by one-carbon metabolism in Escherichia coli.

A genetic screen designed to isolate mutants of Escherichia coli W3110 altered in the ability to induce the heat shock response identified a strain unable to induce the heat shock proteins in a rich, defined medium lacking methionine after exposure to 2,4-dinitrophenol. This strain also grew slowly at 28 degrees C and linearly at 42 degrees C in this medium. The abnormal induction of the heat shock proteins and abnormal growth at both high and low temperatures were reversed when methionine was included in the growth medium. The mutation responsible for these phenotypes mapped to the glyA gene, a biosynthetic gene encoding the enzyme that converts serine and tetrahydrofolate to glycine and 5,10-methylenetetrahydrofolate. This reaction is the major source of glycine and one-carbon units in the cell. Because fixed one-carbon units, in the form of methionine, allowed mutant cells to induce the heat shock response after exposure to 2,4-dinitrophenol, a one-carbon restriction may be responsible for the phenotypes described above.     

The role of SHMT2 in modulating lipid metabolism in hepatocytes via glycine-mediated mTOR activation

Serine hydroxymethyltransferase 2 (SHMT2) converts serine into glycine in the mitochondrial matrix, transferring a methyl group to tetrahydrofolate. SHMT2 plays an important role in the maintenance of one-carbon metabolism. Previously, we found a negative correlation between the serine concentration and the progression of fatty liver disease (FLD). However, little is known about the role of SHMT2 in hepatic lipid metabolism. We established SHMT2 knockdown (KD) mouse primary hepatocytes using RNA interference to investigate the role of SHMT2 in lipid metabolism. SHMT2 KD hepatocytes showed decreased lipid accumulation with reduced glycine levels compared to the scramble cells, which was restored upon reintroducing SHMT2. SHMT2 KD hepatocytes showed downregulation of the mTOR/PPAR? pathway with decreased gene expression related to lipogenesis and fatty acid uptake. Pharmacological activation of mTOR or PPAR? overexpression blocked the inhibitory effect of SHMT2 KD on lipid accumulation. We also showed that glycine activated mTOR/PPAR? signaling and identified glycine as a mediator of SHMT2-responsive lipid accumulation in hepatocytes. In conclusion, silencing SHMT2 in hepatocytes ameliorates lipid accumulation via the glycine-mediated mTOR/PPAR? pathway. Our findings underscore the possibility of SHMT2 as a therapeutic target of FLD.

     

Enzymatic production of L-serine

Serine hydroxymethyltransferase (SHMT) in the form of crude extract form a recombinant strain of Klebsiella aerogenes was used to study the production of L-serine from glycine and formaldehyde (HCHO). SHMT activity linearly increased with temperature (30-50 degrees C). Addition of exogenous cofactors, tetrahydrofolic acid and pyridoxal-phosphate, significantly increased SHMT activity. The pH optimum of the SHMT catalyzed L-serine synthesis step was between 8.0 and 8.5. The K(m) for glycine was 11.6mM at 37 degrees C and pH 8.0. A 87% molar conversion of glycine to serine was obtained at equilibrium (37 degrees C, pH 8.0). Tetrahydrofolic acid was stabilized by maintaining the redox potential of the reaction solution below -330 mV through the addition of a reducing reagent such as beta-mercaptoethanol. SHMT stability was very sensitive to HCHO concentration. By carefully balancing the HCHO feed rate against the enzymatic bioconversion rate in order to keep HCHO concentration low, a serine titer of 160 g/L was achieved, the residual glycine concentration was reduced to 40 g/L, a 70% molar conversion of glycine with quantitative yield was obtained, and the overall serine productivity was 5.2 g/L/h.     

Characterisation of a human serine hydroxymethyltransferase pseudogene and its localisation to 1p32.3–33

The conversion of serine and tetrahydrofolate to glycine and 5,10 methylene tetrahydrofolate by serine hydroxymethyltransferase (SHMT, EC 2.1.2.1) is the major route for the provision of one-carbon units for biosynthetic reactions. SHMT cDNAs have been cloned from both rabbit and man, and a human mitochondrial SHMT gene sequence has recently been reported. We have isolated phage clones containing human genomic sequences homologous to cytosolic SHMT and have found these to contain a processed pseudogene (SHMT-psl) with a 90% identity to cloned SHMT cDNAs. SHMT ps1 contains 2335 nt that are homologous to SHMT but it has an additional leader sequence of 203 nt of unknown origin. The complete SHMT-ps.1 sequence of 2538 nt is bounded by two 16 nt direct repeats that are characteristic of retroelement insertion sites. Two phage clones containing SHMT-ps1 have been mapped by fluorescence in situ hybridisation to 1p32.3–33. ln addition, an SHMT cDNA clone hybridized to the same region and to 17p11.2–12. The latter is consistent with a previous localisation of the gene for cytosolic SHMT.     

glyA基因及其编码的丝氨酸羟甲基转移酶

glyA基因广泛存在于生物体中 ,其编码的丝氨酸羟甲基转移酶 (serinehydroxymethyltransferase,SHMT)催化丝氨酸和甘氨酸之间的相互转化 ,转化反应产生的 5,1 0 亚甲基四氢叶酸 (M THF)提供细胞新陈代谢—碳单位 ,此反应在细胞新陈代谢中处于重要地位。因此 ,研究 glyA基因及其编码的丝氨酸羟甲基转移酶具有重要的意义。介绍了 glyA基因的克隆、序列分析、调控组分和丝氨酸羟甲基转移酶的部分性质。     

Secondary metabolite biosynthesis in cultured cells of Catharanthus roseus (L.) G. Don immobilized by adhesion to glass fibres

Summary Suspension-cultured cells of Catharanthus roseus (L.) G. Don were immobilized on glass fibre mats and cultivated in shake flasks. The highly-aggregated immobilized cells exhibited a slower growth rate and accumulated reduced levels of tryptamine and indole alkaloids, represented by catharanthine and ajmalicine, in comparison to cells in suspension. The increased total protein synthesis in immobilized cells suggests a diversion of the primary metabolic flux toward protein biosynthetic pathways and away from other growth processes. In vitro assays for the specific activity of tryptophan decarboxylase (TDC) and tryptophan synthase (TS) suggest that the decreased accumulation of tryptamine in immobilized cells was due to reduced tryptophan biosynthesis. The specific activity of TDC was similar in immobilized and suspension-cultured cells. However, the expression of TS activity in immobilized cells was reduced to less than 25% of the maximum level in suspension-cultured cells. The reduced availability of a free tryptophan pool in immobilized cells is consistent with the reduced TS activity. Reduced tryptamine accumulation, however, was not responsible for the decreased accumulation of indole alkaloids in immobilized cells. Indole alkaloid accumulation increased to a similar level in immobilized and suspension-cultured cells only after the addition of exogenous secolaganin to the culture medium. The addition of tryptophan resulted in increased accumulation of tryptamine, but had no effect on indole alkaloid levels. Reduced biosynthesis of secologanin, the monoterpenoid precursor to indole alkaloids, in immobilized cells is suggested. Immobilization does not appear to alter the activity of indole alkaloid biosynthetic enzymes in our system beyond, and including, strictosidine synthase. Offprint requests to: P. J. Facchini     

One-carbon metabolism in lectin-activated human lymphocytes

Serine is an essential amino acid for the lectin-mediated transformation of human peripheral blood lymphocytes due to the inability of this cell to synthesize sufficient quantities via either the phosphorylated pathway or by reversal of the serine hydroxymethyltransferase reaction to meet the metabolic demands. The level of intracellular serine is tightly regulated, and the culture medium concentration for optimum cellular transformation falls within a relatively narrow range. The three-carbon atom of serine is the major source of one-carbon units required for purine and pyrimidine nucleotide biosynthesis, but the key effect of both serine deprivation and of high medium serine levels would appear to be on protein synthesis. Although an alternative source of one-carbon units, as provided by high levels of formate in the culture medium, can partially reverse the effects of serine deprivation, the only other demonstrable source of one-carbon units, tryptophan, requires serine for its incorporation and subsequent metabolism. Methionine is also essential for lymphocyte transformation and is involved in the synthesis of a small amount of phosphatidylcholine, although most of this phospholipid is provided by choline and lysophosphatidylcholine from the serum-supplemented culture medium.     

Identification of glyA as a symbiotically essential gene in Bradyrhizobium japonicum

A Bradyrhizobium japonicum Tn5 mutant (strain 3160) induced numerous, tiny, white nodules which were dispersed over the whole root system of its natural host plant, soybean (Glycine max). These ineffective, nitrogen non-fixing pseudonodules were disturbed at a very early step of bacteroid and nodule development. Subsequent cloning and sequencing of the DNA region mutated in strain 3160 revealed that the Tn5 insertion mapped in a gene that had 60% homology to the Escherichia coli glyA gene coding for serine hydroxymethyltransferase (SHMT; E.C.2.1.2.1.). SHMT catalyses the biosynthesis of glycine from serine and the transfer of a one-carbon unit to tetrahydrofolate. The B. japonicum glyA region was able to fully complement the glycine auxotrophy of an E. coli glyA deletion strain. Although the Tn5 insertion in B. japonicum mutant 3160 disrupted the glyA coding sequence, this strain was only a bradytroph (i.e. a leaky auxotroph). Thus, B. japonicum may have an additional pathway for glycine biosynthesis. Nevertheless, the glyA mutation was responsible for the drastic symbiotic phenotype visible on plants. It may be possible, therefore, that a sufficient supply with glycine and/or a functioning C1-metabolism are indispensable for the establishment of a fully effective, nitrogen-fixing root nodule symbiosis.     

Production of l-serine from glycine by Corynebacterium glycinophilum and properties of serine hydroxymethyltransferase,a key enzyme in l-serine production

Fermentative production of l-serine from glycine by Corynebacterium glycinophilum AJ-3413, an auxotrophic mutant of Leu and Met with increased productivity of l-serine using a one liter jar fermentor was carried out and the properties of serine hydroxymethyltransferase (SHMT), a key enzyme in l-serine synthesis, of the parental strain AJ-3170 were investigated. SHMT was effectively induced by the addition of glycine to the medium at an early stage of cultivation. Under optimal conditions, AJ-3413 produced 16.0 g/l of l-serine from 30 g/l of glycine with a molar yield of 38%. The partially purified SHMT catalyzed the l-allo-threonine degradation in addition to l-serine degradation, but could not catalyze l-threonine degradation. This enzyme showed an absolute tetrahydrofolic acid requirement for l-serine degradation to glycine and formaldehyde, but not for l-allo-threonine degradation. Pyridoxal 5′-phosphate appeared to be required for enzyme activity. The K_m values for glycine and formaldehyde in l-serine synthesis, and for l-serine in l-serine degradation were 1.85, 0.29 and 1.64 mM, respectively.     

The glycine decarboxylase complex is not essential for the cyanobacterium Synechocystis sp. strain PCC 6803

In order to investigate the metabolic importance of glycine decarboxylase (GDC) in cyanobacteria, mutants were generated defective in the genes encoding GDC subunits and the serine hydroxymethyl-transferase (SHMT). It was possible to mutate the genes for GDC subunits P, T, or H protein in the cyanobacterial model strain Synechocystis sp. PCC 6803, indicating that GDC is not necessary for cell viability under standard conditions. In contrast, the SHMT coding gene was found to be essential. Almost no changes in growth, pigmentation, or photosynthesis were detected in the GDC subunit mutants, regardless of whether or not they were cultivated at ambient or high CO2 concentrations. The mutation of GDC led to an increased glycine/serine ratio in the mutant cells. Furthermore, supplementation of the medium with low glycine concentrations was toxic for the mutants but not for wild type cells. Conditions stimulating photorespiration in plants, such as low CO2 concentrations, did not induce but decrease the expression of the GDC and SHMT genes in Synechocystis. It appears that, in contrast to heterotrophic bacteria and plants, GDC is dispensable for Synechocystis and possibly other cyanobacteria.     

Serine hydroxymethyltransferase: role of glu75 and evidence that serine is cleaved by a retroaldol mechanism

Serine hydroxymethyltransferase (SHMT) catalyzes the reversible interconversion of serine and glycine with tetrahydrofolate serving as the one-carbon carrier. SHMT also catalyzes the folate-independent retroaldol cleavage of allothreonine and 3-phenylserine and the irreversible conversion of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate. Studies of wild-type and site mutants of SHMT have failed to clearly establish the mechanism of this enzyme. The cleavage of 3-hydroxy amino acids to glycine and an aldehyde occurs by a retroaldol mechanism. However, the folate-dependent cleavage of serine can be described by either the same retroaldol mechanism with formaldehyde as an enzyme-bound intermediate or by a nucleophilic displacement mechanism in which N5 of tetrahydrofolate displaces the C3 hydroxyl of serine, forming a covalent intermediate. Glu75 of SHMT is clearly involved in the reaction mechanism; it is within hydrogen bonding distance of the hydroxyl group of serine and the formyl group of 5-formyltetrahydrofolate in complexes of these species with SHMT. This residue was changed to Leu and Gln, and the structures, kinetics, and spectral properties of the site mutants were determined. Neither mutation significantly changed the structure of SHMT, the spectral properties of its complexes, or the kinetics of the retroaldol cleavage of allothreonine and 3-phenylserine. However, both mutations blocked the folate-dependent serine-to-glycine reaction and the conversion of methenyltetrahydrofolate to 5-formyltetrahydrofolate. These results clearly indicate that interaction of Glu75 with folate is required for folate-dependent reactions catalyzed by SHMT. Moreover, we can now propose a promising modification to the retroaldol mechanism for serine cleavage. As the first step, N5 of tetrahydrofolate makes a nucleophilic attack on C3 of serine, breaking the C2-C3 bond to form N5-hydroxymethylenetetrahydrofolate and an enzyme-bound glycine anion. The transient formation of formaldehyde as an intermediate is possible, but not required. This mechanism explains the greatly enhanced rate of serine cleavage in the presence of folate, and avoids some serious difficulties presented by the nucleophilic displacement mechanism involving breakage of the C3-OH bond.     

Tryptophan Synthase and Production of l-Tryptophan in Regulatory Mutants

A 5-fluorotryptophan-resistant mutant of Brevibacterium flavum, No. 187, accumulated 2.6 g of indole 3-glycerol (InG) in addition to 8.0 g of l-tryptophan per liter in the culture medium. The addition of l-serine to the medium decreased the accumulation of InG and increased that of l-tryptophan up to a concentration of 10.3 g/liter, while the addition of l-tryptophan increased the InG accumulation, suggesting that InG was formed by hydrolysis of indole 3-glycerol phosphate (InGP), the substrate of tryptophan synthase (TS) which catalyzed the final step reaction of tryptophan biosynthesis. Then, in order to examine the mechanism of the InG accumulation, TS was purified from tryptophan auxotroph, TA-60. The reaction mechanism of TS was Ordered Bi Bi with Km’s of 0.63 and 0.038 mm for serine and InGP, respectively. Tryptophan, a product of the TS reaction, inhibited TS competitively with respect to serine and the Ki for tryptophan was estimated to be 2.0 mm. On the other hand, anthranilate synthase (AS), the first enzyme in the tryptophan biosynthetic pathway, was much less sensitive to the feedback inhibition by tryptophan in strain No. 187 than in the wild strain. The tryptophan concentration giving 50% inhibition of AS in strain No. 187 was estimated to be 2.4 mm, almost comparable to that of TS, 7.7 mm. From these results, it was concluded that the accumulation of InG in strain No. 187 would result from the product inhibition of TS by the tryptophan accumulated.     

Glycine and serine catabolism in non-photosynthetic higher plant cells: their role in C1 metabolism

Glycine and serine are two interconvertible amino acids that play an important role in C1 metabolism. Using 13C NMR and various 13C-labelled substrates, we studied the catabolism of each of these amino acids in non-photosynthetic sycamore cambial cells. On one hand, we observed a rapid glycine catabolism that involved glycine oxidation by the mitochondrial glycine decarboxylase (GDC) system. The methylenetetra- hydrofolate (CH2-THF) produced during this reaction did not equilibrate with the overall CH2-THF pool, but was almost totally recycled by the mitochondrial serine hydroxymethyltransferase (SHMT) for the synthesis of one serine from a second molecule of glycine. Glycine, in contrast to serine, was a poor source of C1 units for the synthesis of methionine. On the other hand, catabolism of serine was about three times lower than catabolism of glycine. Part of this catabolism presumably involved the glycolytic pathway. However, the largest part (about two-thirds) involved serine-to-glycine conversion by cytosolic SHMT, then glycine oxidation by GDC. The availability of cytosolic THF for the initial SHMT reaction is possibly the limiting factor of this catabolic pathway. These data support the view that serine catabolism in plants is essentially connected to C1 metabolism. The glycine formed during this process is rapidly oxidized by the mitochondrial GDC-SHMT enzymatic system, which is therefore required in all plant tissues.