Ser3p (Yer081wp) and Ser33p (Yil074cp) are phosphoglycerate dehydrogenases in Saccharomyces cerevisiae

Two genes YER081W and YIL074C, renamed SER3 and SER33, respectively, which encode phosphoglycerate dehydrogenases in Saccharomyces cerevisiae were identified. These dehydrogenases catalyze the first reaction of serine and glycine biosynthesis from the glycolytic metabolite 3-phosphoglycerate. Unlike either single mutant, the ser3Delta ser33Delta double mutant lacks detectable phosphoglycerate dehydrogenase activity and is auxotrophic for serine or glycine for growth on glucose media. However, the requirement for the SER-dependent "phosphoglycerate pathway" is conditional since the "glyoxylate" route of serine/glycine biosynthesis is glucose-repressed. Thus, in cells grown on ethanol both expression and activity of all SER-encoded proteins are low, including the remaining enzymes of the phosphoglycerate pathway, Ser1p and Ser2p. Moreover the available nitrogen source regulates the expression of SER genes. However, for only SER33, and not SER3, expression was regulated in relation to the available nitrogen source in a coordinated fashion with SER1 and SER2. Based on these mRNA data together with data on enzyme activities, Ser33p is likely to be the main isoenzyme of the phosphoglycerate pathway during growth on glucose. Moreover, since phosphoglycerate dehydrogenase activity requires NAD(+) as cofactor, deletion of SER3 and SER33 markedly affected redox metabolism as shown by substrate and product analysis.     

Mutational analysis of serine-glycine biosynthesis in Rhodopseudomonas capsulata.

Rhodopseudomonas capsulata possesses the enzymes of both the "phosphorylated" and the "non-phosphorylated" pathways of serine biosynthesis. Certain mutants with lesions in the phosphorylated pathway are serine-glycine auxotrophs, though they still produce enzymes of the non-phosphorylated sequence. These results indicate that the phosphorylated pathway is essential for the synthesis of serine and glycine in R. capsulata under the condtions tested.     

"Active" one-carbon generation in Saccharomyces cerevisiae.

A new mutation introducing a one-carbon requirement (e.g., formate) for the glycine-supplemented growth of a serine-glycine auxotroph (ser1) was correlated with a lack of glycine decarboxylase activity. The presence of oxalate decarboxylase activity or glyoxylate decarboxylase activity did not overcome the one-carbon requirement. Another mutation characterized by the absence of oxalate decarboxylase activity did not introduce a one-carbon requirement. The presence and physiological significance of glycine decarboxylase activity in Saccharomyces are thus inferred.     

Biosynthesis of glyoxylate from glycine in Saccharomyces cerevisiae

Glyoxylate biosynthesis in Saccharomyces cerevisiae is traditionally mainly ascribed to the reaction catalyzed by isocitrate lyase (Icl), which converts isocitrate to glyoxylate and succinate. However, Icl is generally reported to be repressed by glucose and yet glyoxylate is detected at high levels in S. cerevisiae extracts during cultivation on glucose. In bacteria there is an alternative pathway for glyoxylate biosynthesis that involves a direct oxidation of glycine. Therefore, we investigated the glycine metabolism in S. cerevisiae coupling metabolomics data and (13)C-isotope-labeling analysis of two reference strains and a mutant with a deletion in a gene encoding an alanine:glyoxylate aminotransferase. The strains were cultivated on minimal medium containing glucose or galactose, and (13)C-glycine as sole nitrogen source. Glyoxylate presented (13)C-labeling in all cultivation conditions. Furthermore, glyoxylate seemed to be converted to 2-oxovalerate, an unusual metabolite in S. cerevisiae. 2-Oxovalerate can possibly be converted to 2-oxoisovalerate, a key precursor in the biosynthesis of branched-chain amino acids. Hence, we propose a new pathway for glycine catabolism and glyoxylate biosynthesis in S. cerevisiae that seems not to be repressed by glucose and is active under both aerobic and anaerobic conditions. This work demonstrates the great potential of coupling metabolomics data and isotope-labeling analysis for pathway reconstructions.     

Disruption of cytoplasmic and mitochondrial folylpolyglutamate synthetase activity in Saccharomyces cerevisiae

Similar to other eukaryotes, yeasts have parallel pathways of one-carbon metabolism in the cytoplasm and mitochondria and have folylpolyglutamate synthetase activity in both compartments. The gene encoding folylpolyglutamate synthetase is MET7 (also referred to as MET23) on chromosome XV and appears to encode both the cytoplasmic and mitochondrial forms of the enzyme. In order to determine the metabolic roles of both forms of folylpolyglutamate synthetase, we disrupted the met7 gene and determined that the strain is a methionine auxotroph and an adenine and thymidine auxotroph when grown in the presence of sulfanilamide. The met7 mutant becomes petite under normal growth conditions but can be maintained with a grande phenotype if the strain is tup and all media are supplemented with dTMP. A met7 gly1 strain is auxotrophic for glycine when grown on glucose but prototrophic when grown on glycerol. A met7 ser1 strain cannot use glycine to suppress the serine auxotrophy of the ser1 phenotype. A met7 shm2 strain is nonviable. In order to disrupt just the mitochondrial folylpolyglutamate synthetase activity, we constructed mutants with an inactivated chromosomal MET7 gene complemented by genes that express only cytoplasmic folylpolyglutamate synthetase, including the Lactobacillus casei folC gene and the yeast MET7 gene with its mitochondrial leader sequence deleted (MET7Deltam). All the genes providing cytoplasmic folylpolyglutamate synthetase complemented the methionine auxotrophy as well as the synthetic lethality of the shm2 strain and the synthetic glycine auxotrophy of the gly1 strain. The strains lacking the mitochondrial folylpolyglutamate synthetase had longer doubling times than the isogenic wild-type strains but retained the function of the mitochondrial folate-dependent enzymes to produce formate, serine, and glycine. Mutants complemented by the bacterial folC gene or by the MET7Deltam gene on a 2mu plasmid remained grande without the tup mutation and supplementation and dTMP. Mutants complemented by the MET7Deltam gene integrated in single copy became petites under those conditions, indicating a deficiency in dTMP production but this is likely due to lower expression of cytoplasmic folylpolyglutamate synthetase by the MET7Deltam gene.     

Acetate utilization by a methylotrophic bacterium

The mode of acetate ultilization in the Gram-negative diplococcoid bacterium PAR was investigated. This organism uses the isocitrate lyase-negative serine pathway during growth on C₁-compounds and was found to lack isocitrate lyase on acetate growth also. Enzyme assays revealed the absence of the glycerate and hydroxyaspartate pathways for the metabolism of C₂-compounds. Pulse-labeling experiments indicated the rapid formation of glycine, glutamate, and malate. The rapid formation of malate and the presence of malate synthase suggest that glyoxylate is an intermediate formed from acetate by a means other than the conventional isocitrate cleavage reaction.     

Flux of the L-serine metabolism in rat liver. The predominant contribution of serine dehydratase.

L-Serine metabolism in rat liver was investigated, focusing on the relative contributions of the three pathways, one initiated by L-serine dehydratase (SDH), another by serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT), and the other involving serine hydroxymethyltransferase and the mitochondrial glycine cleavage enzyme system (GCS). Because serine hydroxymethyltransferase is responsible for the interconversion between serine and glycine, SDH, SPT/AGT, and GCS were considered to be the metabolic exits of the serine-glycine pool. In vitro, flux through SDH was predominant in both 24-h starved and glucagon-treated rats. Flux through SPT/AGT was enhanced by glucagon administration, but even after the induction, its contribution under quasi-physiological conditions (1 mM L-serine and 0.25 mM pyruvate) was about (1)/(10) of that through SDH. Flux through GCS accounted for only several percent of the amount of L-serine metabolized. Relative contributions of SDH and SPT/AGT to gluconeogenesis from L-serine were evaluated in vivo based on the principle that 3H at the 3 position of L-serine is mostly removed in the SDH pathway, whereas it is largely retained in the SPT/AGT pathway. The results showed that SPT/AGT contributed only 10-20% even after the enhancement of its activity by glucagon. These results suggested that SDH is the major metabolic exit of L-serine in rat liver.     

Glyoxylate regeneration pathway in the methylotroph Methylobacterium extorquens AM1

Most serine cycle methylotrophic bacteria lack isocitrate lyase and convert acetyl coenzyme A (acetyl-CoA) to glyoxylate via a novel pathway thought to involve butyryl-CoA and propionyl-CoA as intermediates. In this study we have used a genome analysis approach followed by mutation to test a number of genes for involvement in this novel pathway. We show that methylmalonyl-CoA mutase, an R-specific crotonase, isobutyryl-CoA dehydrogenase, and a GTPase are involved in glyoxylate regeneration. We also monitored the fate of (14)C-labeled carbon originating from acetate, butyrate, or bicarbonate in mutants defective in glyoxylate regeneration and identified new potential intermediates in the pathway: ethylmalonyl-CoA, methylsuccinyl-CoA, isobutyryl-CoA, methacrylyl-CoA, and beta-hydroxyisobutyryl-CoA. A new scheme for the pathway is proposed based on these data.     

Metabolic flux analysis for a ppc mutant Escherichia coli based on 13C-labelling experiments together with enzyme activity assays and intracellular metabolite measurements

The physiology and central metabolism of a ppc mutant Escherichia coli were investigated based on the metabolic flux distribution obtained by (13)C-labelling experiments using gas chromatography-mass spectrometry (GC-MS) and 2-dimensional nuclear magnetic resonance (2D NMR) strategies together with enzyme activity assays and intracellular metabolite concentration measurements. Compared to the wild type, its ppc mutant excreted little acetate and produced less carbon dioxide at the expense of a slower growth rate and a lower glucose uptake rate. Consequently, an improvement of the biomass yield on glucose was observed in the ppc mutant. Enzyme activity measurements revealed that isocitrate lyase activity increased by more than 3-fold in the ppc mutant. Some TCA cycle enzymes such as citrate synthase, aconitase and malate dehydrogenase were also upregulated, but enzymes of glycolysis and the pentose phosphate pathway were downregulated. The intracellular intermediates in the glycolysis and the pentose phosphate pathway, therefore, accumulated, while acetyl coenzyme A and oxaloacetate concentrations decreased in the ppc mutant. The intracellular metabolic flux analysis uncovered that deletion of ppc resulted in the appearance of the glyoxylate shunt, with 18.9% of the carbon flux being channeled via the glyoxylate shunt. However, the flux of the pentose phosphate pathway significantly decreased in the ppc mutant.     

Aspects of glycine and serine biosynthesis during growth of Pseudomonas AM1 on C1 compounds

1. Methanol or formate can replace serine or glycine as supplements for growth on succinate of the auxotrophic mutants 20S and 82G of Pseudomonas AM1, showing that the organism can synthesize glycine and serine in net fashion from C(1) units. 2. Double mutants of Pseudomonas 20S and 82G have been prepared (20ST-1 and 82GT-1) that are unable to grow on succinate+1mm-glyoxylate, succinate+2mm-methanol or methanol alone. 3. Mutants 20ST-1 and 82GT-1 lacked serine-glyoxylate aminotransferase activity, and revertants to the phenotype of 20S and 82G regained serine-glyoxylate aminotransferase activity. A total revertant of 82GT-1 to wild-type phenotype regained activities of serine hydroxymethyltransferase and serine-glyoxylate aminotransferase. 4. The activity of serine-glyoxylate aminotransferase in methanol-grown Pseudomonas AM1 is eightfold higher than in the succinate-grown organism. 5. The combined results show that in Pseudomonas AM1 serine-glyoxylate aminotransferase is necessary for growth on C(1) compounds and is involved in the conversion of methanol into glycine via glyoxylate. 6. It is suggested that the phosphorylated pathway of serine biosynthesis from phosphoglycerate replenishes the supply of alpha-amino groups necessary for the flow of glyoxylate through the main assimilatory pathway during growth on C(1) compounds.     

基于代谢流量分布信息理解大肠杆菌中异柠檬酸裂解酶调节因子的代谢调控作用

基因的表达受不同的转录调节因子调节。大肠杆菌中的异柠檬酸裂解酶调节因子(IclR)能够抑制编码乙醛酸支路酶的aceBAK操纵子的表达。本研究基于代谢物的13C同位体物质分布来定量解析代谢反应,主要研究了iclR基因在大肠杆菌生理和代谢中的作用。大肠杆菌iclR基因缺失突变株的生长速率、糖耗速率和乙酸的产量相对于原始菌株都有所降低,但菌体得率略有增加。通过代谢途径的流量比率分析发现基因缺失株的乙醛酸支路得到了激活,33%的异柠檬酸流经了乙醛酸支路;戊糖磷酸途径的流量变小,使得CO2的生成量减少。同时,乙醛酸支路激活,但草酰乙酸形成磷酸烯醇式丙酮酸的流量基本不变,说明磷酸烯醇式丙酮酸-乙醛酸循环没有激活,没有过多的碳原子在磷酸烯醇式丙酮酸羧化激酶反应中以CO2形式排出,从而确保了菌体得率。葡萄糖利用速率的降低、乙酰辅酶A的代谢效率提高等使得iclR基因敲除菌的乙酸分泌较原始菌株有所降低。     

Metabolic Role, Regulation of Synthesis, Cellular Localization, and Genetic Control of the Glyoxylate Cycle Enzymes in Neurospora crassa

The glyoxylate shunt enzymes, isocitrate lyase and malate synthase, were present at high levels in mycelium grown on acetate as sole source of carbon, compared with mycelium grown on sucrose medium. The glyoxylate shunt activities were also elevated in mycelium grown on glutamate or Casamino Acids as sole source of carbon, and in amino acid-requiring auxotrophic mutants grown in sucrose medium containing limiting amounts of their required amino acid. Under conditions of enhanced catabolite repression in mutants grown in sucrose medium but starved of Krebs cycle intermediates, isocitrate lyase and malate synthase levels were derepressed compared with the levels in wild type grown on sucrose medium. This derepression did not occur in related mutants in which Krebs cycle intermediates were limiting growth but catabolite repression was not enhanced. No Krebs cycle intermediate tested produced an efficient repression of isocitrate lyase activity in acetate medium. Of the two forms of isocitrate lyase in Neurospora, isocitrate lyase-1 constituted over 80% of the isocitrate lyase activity in acetate-grown wild type and also in each of the cases already outlined in which the glyoxylate shunt activities were elevated on sucrose medium. On the basis of these results, it is concluded that the synthesis of isocitrate lyase-1 and malate synthase in Neurospora is regulated by a glycolytic intermediate or derivative. Our data suggest that isocitrate lyase-1 and isocitrate lyase-2 are the products of different structural genes. The metabolic roles of the two forms of isocitrate lyase and of the glyoxylate cycle are discussed on the basis of their metabolic control and intracellular localization.     

Acetate metabolism in Rhodopseudomonas gelatinosa and several other Rhodospirillaceae

When Rhodopseudomonas gelatinosa was grown on acetate aerobically in the dark both enzymes of the glyoxylate bypass, isocitrate lyase and malate synthase, could be detected. However, under anaerobic conditions in the light only isocitrate lyase, but not malate synthase, could be found.The reactions, which bypass the malate synthase reaction are those catalyzed by alanine glyoxylate aminotransferase and the enzymes of the serine pathway.Other Rhodospirillaceae were tested for isocitrate lyase and malate synthase activity after growth with acetate; they could be divided into three groups: I. organisms possessing both enzymes; 2. organisms containing malate synthase only; 3. R. gelatinosa containing only isocitrate lyase when grown anaerobically in the light.     

Regulation of phosphoglycerate dehydrogenase levels and effect on serine synthesis in Escherichia coli K-12.

The level of phosphoglycerate dehydrogenase, the first enzyme in the biosynthetic pathway to serine and glycine, has been studied in Escherichia coli grown under different conditions. The enzyme level was not reduced by growth in a medium which contained the end products of the pathway, nor was it elevated when the growth rates was limited by the supply of serine. Elevation of phosphoglycerate dehydrogenase did not occur when charging of tRNA ser was inhibited by serine hydroxamate. However, phosphoglycerate dehydrogenase levels were subject to regulation. Elevated levels of enzyme activity were observed in merodiploids containing multiple copies of the serA gene, and lowered enzyme levels were found in cells grown on carbon sources other than glucose or when certain amino acids were present in the growth medium. The combined effect of these nutritional changes (carbon source and amino acids) reduced the level of phosphoglycerate dehydrogenase to 10 to 12% of that found in wild-type cells and to about 5% of the level in the merodiploids. By using antibody prepared against purified phosphoglycerate dehydrogenase we established that the decrease in enzyme activity reflected decreased amounts of enzyme protein. Constant intracellular concentrations of 3-phosphoglycerate and serine were found in cells with marked differences in phosphoglycerate dehydrogenase activity, indicating that end product inhibition of phosphoglycerate dehydrogenase activity, rather than the amount of the biosynthetic enzymes, is the major factor in regulating the intracellular concentration of serine.     

Unique glycine-activated riboswitch linked to glycine–serine auxotrophy in SAR11

The genome sequence of the marine bacterium ' Candidatus Pelagibacter ubique' and subsequent analyses have shown that while it has a genome as small as many obligate parasites, it nonetheless possesses a metabolic repertoire that allows it to grow as one of the most successful free-living cells in the ocean. An early report based on metabolic reconstruction indicated that SAR11 cells are prototrophs for all amino acids. However, here we report experimental evidence that ' Cand. P. ubique' is effectively auxotrophic for glycine and serine. With glucose and acetate added to seawater to supply organic carbon, the addition of 125 nM to 1.5 μM glycine to growth medium containing all other nutrients in excess resulted in a linear increase in maximum cell density from 1.14 × 10⁶ cells ml⁻¹ to 8.16 × 10⁶ cells ml⁻¹ ( R ² =  0.992). Serine was capable of substituting for glycine at 1.5 μM. ' Cand. P. ubique' contains a glycine-activated riboswitch preceding malate synthase, an unusual genomic context that is conserved in the SAR11 group. Malate synthase plays a critical role in central metabolism by enabling TCA intermediates to be regenerated through the glyoxylate cycle. In vitro analysis of this riboswitch indicated that it responds solely to glycine but not close structural analogues, such as glycine betaine, malate, glyoxylate, glycolate, alanine, serine or threonine. We conclude that ' Cand. P. ubique' is therefore a glycine–serine auxotroph that appears to use intracellular glycine level to regulate its use of carbon for biosynthesis and energy. Comparative genomics and metagenomics indicate that these conclusions may hold throughout much of the SAR11 clade.     

Purification and characterization of Acinetobacter calcoaceticus isocitrate lyase.

Acinetobacter calcoaceticus is capable of growing on acetate or compounds that are metabolized to acetate. During adaptation to growth on acetate, A. calcoaceticus B4 exhibits an increase in NADP(+)-isocitrate dehydrogenase and isocitrate lyase activities. In contrast, during adaptation to growth on acetate, Escherichia coli exhibits a decrease in NADP(+)-isocitrate dehydrogenase activity that is caused by reversible phosphorylation of specific serine residues on this enzyme. Also, in E. coli, isocitrate lyase is believed to be active only in the phosphorylated form. This phosphorylation of isocitrate lyase may regulate entry of isocitrate into the glyoxylate bypass. To understand the relationships between these two isocitrate-metabolizing enzymes and the metabolism of acetate in A. calcoaceticus B4 better, we have purified isocitrate lyase to homogeneity. Physical and kinetic characterization of the enzyme as well as the inhibitor specificity and divalent cation requirement have been examined.     

Studies of Glycollate Utilization and Some Associated Enzymes of C1 Metabolism in the Endosperm of Ricinus communis L.

Glycollate metabolism in 5-day-old endosperm tissues of Ricinuscommunis L. was examined by feeding micromolar quantities of[2-¹⁴C]glycollate to tissue slices. It was found that glycollatecarbon was rapidly incorporated into glyoxylate, glycine, serine,and carbon dioxide. Only small amounts of ¹⁴C were incorporatedinto the sugars. Changes in the distribution of ¹⁴C with timesuggested that glyoxylate was a primary product and that glycineand serine were secondary products of glycollate metabolism.The results of feeding experiments are interpreted as indicatingthat a glycollate pathway leading to sugar biosynthesis is ofminor importance compared to the rapid utilization of glycollatefor the biosynthesis of glycine and serine. Enzymes necessaryto catalyse the incorporation of glycollate into glycine andserine have been examined in castor-bean endosperm extracts.These included: glycollic acid oxidase, gloxylic acid reductase,glyoxylate transaminase, N¹⁰ formyltetrahydrofolate synthetase,N⁵,N¹⁰-methylenetetrahydrofolate dehydrogenase, and serine hydroxymethyltransferase.     

The isocitrate dehydrogenase phosphorylation cycle: Regulation and enzymology

Isocitrate dehydrogenase (IDH) of Escherichia coli is regulated by phosphorylation and dephosphorylation. This phosphorylation cycle controls the flow of isocitrate through the glyoxylate bypass, a pathway which bypasses the CO₂ evolving steps of the Krebs' cycle. IDH is phosphorylated at a single serine which resides in its active site. Phosphorylation blocks isocitrate binding, thereby inactivating IDH. The IDH phosphorylation cycle is catalyzed by a bifunctional protein kinase/phosphatase. The kinase and phosphatase reactions appear to be catalyzed at the same site and may share some catalytic steps. A variety of approaches have been used to examine the IDH phosphorylation cycle in the intact organism. The picture which has emerged is one of an exquisitely sensitive and flexible system which is capable of adapting efficiently to the environment both inside and outside the cell. © 1993 Wiley-Liss, Inc.     

Anaerobic central metabolic pathways in Shewanella oneidensis MR-1 reinterpreted in the light of isotopic metabolite labeling

It has been proposed that during growth under anaerobic or oxygen-limited conditions, Shewanella oneidensis MR-1 uses the serine-isocitrate lyase pathway common to many methylotrophic anaerobes, in which formaldehyde produced from pyruvate is condensed with glycine to form serine. The serine is then transformed through hydroxypyruvate and glycerate to enter central metabolism at phosphoglycerate. To examine its use of the serine-isocitrate lyase pathway under anaerobic conditions, we grew S. oneidensis MR-1 on [1-13C]lactate as the sole carbon source, with either trimethylamine N-oxide (TMAO) or fumarate as an electron acceptor. Analysis of cellular metabolites indicated that a large percentage (>70%) of lactate was partially oxidized to either acetate or pyruvate. The 13C isotope distributions in amino acids and other key metabolites indicate that under anaerobic conditions, although glyoxylate synthesized from the isocitrate lyase reaction can be converted to glycine, a complete serine-isocitrate pathway is not present and serine/glycine is, in fact, oxidized via a highly reversible degradation pathway. The labeling data also suggest significant activity in the anapleurotic (malic enzyme and phosphoenolpyruvate carboxylase) reactions. Although the tricarboxylic acid (TCA) cycle is often observed to be incomplete in many other anaerobes (absence of 2-oxoglutarate dehydrogenase activity), isotopic labeling supports the existence of a complete TCA cycle in S. oneidensis MR-1 under certain anaerobic conditions, e.g., TMAO-reducing conditions.     

Biosynthesis of amino acids in Clostridium pasteurianum

1. Clostridium pasteurianum was grown on a synthetic medium with the following carbon sources: (a) (14)C-labelled glucose, alone or with unlabelled aspartate or glutamate, or (b) unlabelled glucose plus (14)C-labelled aspartate, glutamate, threonine, serine or glycine. The incorporation of (14)C into the amino acids of the cell protein was examined. 2. In both series of experiments carbon from exogenous glutamate was incorporated into proline and arginine; carbon from aspartate was incorporated into glutamate, proline, arginine, lysine, methionine, threonine, isoleucine, glycine and serine. Incorporations from the other exogenous amino acids indicated the metabolic sequence: aspartate --> threonine --> glycine right harpoon over left harpoon serine. 3. The following activities were demonstrated in cell-free extracts of the organism: (a) the formation of aspartate by carboxylation of phosphoenolpyruvate or pyruvate, followed by transamination; (b) the individual reactions of the tricarboxylic acid route to 2-oxoglutarate from oxaloacetate; glutamate dehydrogenase was not detected; (c) the conversion of aspartate into threonine via homoserine; (d) the conversion of threonine into glycine by a constitutive threonine aldolase; (e) serine transaminase, phosphoserine transaminase, glycerate dehydrogenase and phosphoglycerate dehydrogenase. This last activity was abnormally high. 4. The combined evidence indicates that in C. pasteurianum the biosynthetic role of aspartate and glutamate is generally similar to that in aerobic and facultatively aerobic organisms, but that glycine is synthesized from glucose via aspartate and threonine.