Glutamate and serine as competing donors for amination of glyoxylate in leaf peroxisomes

When provided with glycollate, peroxisomal extracts of leaves of spinach beet (Beta vulgaris L. cv.) converted L-serine and L-glutamate to hydroxypyruvate and 2-oxoglutarate respectively. When approximately saturating concentrations of each of these amino acids were incubated separately with glycollate, the utilization of serine was greater than that of glutamate. The utilization of glutamate was substantially reduced by the presence of relatively low concentrations of serine in the reaction mixture, whereas even high concentrations of glutamate caused only small reductions in serine utilization. Over the entire range of concentrations of amino acids examined, serine was invariably the preferred amino-group donor, but this preference was abolished at higher concentrations of glyoxylate. Serine not only competed favourably for glyoxylate but also inhibited L-glutamate: glyoxylate aminotransferase (GGAT), the degree of inhibition depending upon the glyoxylate concentration. Studies of L-serine: glyoxylate aminotransferase (SGAT) and GGAT in partially purified extracts from spinach-beet leaves confirmed that serine competitively inhibited GGAT but glutamate did not affect SGAT. Both enzymes were inhibited by high glyoxylate concentrations, the inhibition being relieved by suitably high concentrations of the appropriate amino acid. It is concluded that at the low glyoxylate concentrations likely to occur in vivo, the preferential utilization of serine would ensure flux through the glycollate pathway to glycerate, but at higher concentrations of glyoxylate, both enzymes could be fully active in glyoxylate amination.Abbreviations SGAT L-serine: glyoxylate aminotransferase - GGAT L-glutamate: glyoxylate aminotransferase     

光呼吸途径中两种转氨酶的研究进展

植物丝氨酸：乙醛酸氨基转移酶（SGAT）与谷氨酸：乙醛酸氨基转移酶（GGAT）主要催乙醛酸的转氨反应,是光呼吸途径中的两种关键酶。此两种酶大都为二聚体,在高等植物体内主要位于过氧化物酶体内,而在真核藻类植物体内则位于线粒体内,对植物的生长发育与抗逆性具有重要影响。本文对SGAT与GGAT在植物光合作用、氨基酸代谢和抗逆性等方面的研究进展进行了综述,以期对SGAT与GGAT的研究有所帮助。     

Identification of photorespiratory glutamate:glyoxylate aminotransferase (GGAT) gene in Arabidopsis

In the photorespiratory process, peroxisomal glutamate:glyoxylate aminotransferase (GGAT) catalyzes the reaction of glutamate and glyoxylate to 2-oxoglutarate and glycine. Although GGAT has been assumed to play important roles for the transamination in photorespiratory carbon cycles, the gene encoding GGAT has not been identified. Here, we report that an alanine:2-oxoglutarate aminotransferase (AOAT)-like protein functions as GGAT in peroxisomes. Arabidopsis has four genes encoding AOAT-like proteins and two of them (namely AOAT1 and AOAT2) contain peroxisomal targeting signal 1 (PTS1). The expression analysis of mRNA encoding AOATs and EST information suggested that AOAT1 was the major protein in green leaves. When AOAT1 fused to green fluorescent protein (GFP) was expressed in BY-2 cells, it was found to be localized to peroxisomes depending on PTS1. By screening of Arabidopsis T-DNA insertion lines, an AOAT1 knockout line (aoat1-1) was isolated. The activity of GGAT and alanine:glyoxylate aminotransferase (AGAT) in the above-ground tissues of aoat1-1 was reduced drastically and, AOAT and glutamate:pyruvate aminotransferase (GPAT) activity also decreased. Peroxisomal GGAT was detected in the wild type but not in aoat1-1. The growth rate was repressed in aoat1-1 grown under high irradiation or without sugar, though differences were slight in aoat1-1 grown under low irradiation, high-CO2 (0.3%) or high-sugar (3% sucrose) conditions. These phenotypes resembled those of photorespiration-deficient mutants. Glutamate levels increased and serine levels decreased in aoat1-1 grown in normal air conditions. Based on these results, it was concluded that AOAT1 is targeted to peroxisomes, functions as a photorespiratory GGAT, plays a markedly important role for plant growth and the metabolism of amino acids.     

On the possibility of involvement of glutamate:glyoxylate and serine:glyoxylate aminotransferases from rye (Secale cereale L.) seedlings in the metabolism of tetrapyrrole compounds.

The activity of highly purified L-serine:glyoxylate aminotransferase (SGAT, EC 2.6.1.45) from rye seedlings was inhibited competitively by 5-aminolevulinate (ALA, Ki = 5 mM) SGAT was activated by hematin. Protoporphyrin IX and hematin inhibited irreversibly the activity of highly purified glutamate:glyoxylate aminotransferase (GGAT, EC 2.6.1.2) from rye seedlings. SGAT was found to catalyse transamination between ALA and hydroxypyruvate, whereas GGAT that between ALA and 2-oxoglutarate or pyruvate. It is suggested that SGAT is involved in the process of degradation of the excess ALA which has not been incorporated into porphyrin compounds.     

The value of mutants unable to carry out photorespiration

Manipulation of the CO₂ concentration of the atmosphere allows the selection of photorespiratory mutants from populations of seeds treated with powerful mutagens such as sodium azide. So far, barley lines deficient in activity of phosphoglycolate phosphatase, catalase, the glycine to serine conversion, glutamine synthetase, glutamate synthase, 2-oxoglutarate uptake and serine: glyoxylate aminotransferase have been isolated. In addition one line of pea lacking glutamate synthase activity and one barley line containing reduced levels of Rubisco are available. The characteristics of these mutations are described and compared with similar mutants isolated from populations of Arabidopsis. As yet, no mutant lacking glutamine synthetase activity has been isolated from Arabidopsis and possible reasons for this difference between barley and Arabidopsis are discussed. The value of these mutant plants in the elucidation of the mechanism of photorespiration and its relationships with CO₂ fixation and amino acid metabolism are highlighted.Abbreviations GS cytoplasmic glutamine synthetase - GS₂ chloroplastic glutamine synthetase - PFR Photon fluence rate - Rubisco Ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP Ribulose-1,5-bisphosphate - SGAT serine:glyoxylate aminotransferase     

Short communication. Serine: glyoxylate aminotransferase exerts no control on photosynthesis

A barley (Hordeum vulgare L.) mutant deficient in serine:glyoxylate aminotransferase (SGAT) was crossed with wild-type plants to generate heterozygous mutants. Plants of the F2 generation with reduced SGAT activities (45-60% of wild-type activities) contained proportionally less SGAT protein. Reduced SGAT activities resulted in the accumulation of serine and, to a smaller extent, of glycine, indicating that the flux through the photorespiratory pathway was restricted. Rates of photosynthesis were, however, not affected by the reduction in SGAT activity.     

Photorespiratory nitrogen cycle – A critical evaluation

The photorespiratory nitrogen cycle proposed by Keys et al. (Nature 275: 741–743, 1978) involved formation of glycine by transamination of glyoxylate in the peroxisomes utilizing glutamate. Subsequently, glycine is oxidized to ammonia, serine and CO₂ in the mitochondria. The ammonia is reassimilated via the GS/GOGAT pathway generating glutamate. In this article, experimental evidence which suggests the occurrence of alternative mechanisms of glycolate and serine synthesis as well as of CO₂ and ammonia evolution is discussed. The problem of utilization of NADH coupled to ATP synthesis during photosynthesis is still unresolved, which complicates the glycine oxidation reaction in light. Further, factors are presented that determine the availability of amino donors in the peroxisomes and of amino acids viz., glycine, serine and glutamate for the operation of the photorespiratory N cycle. Recent evidence regarding the role of formate arising out of the reaction of glyoxylate with H₂O₂ in the regulation of photosynthetic electron flow in the Hill reaction, as well as of photorespiratory substrates functioning as carbon sources for the citric acid cycle in the light or for export to the growing tissues, suggests that the role of photo-respiration in plant metabolism needs to be reexamined.     

Control of photosynthesis in barley mutants with reduced activities of glutamine synthetase and glutamate synthase

Heterozygous mutants of barley (Hordeum vulgare L. cv. Maris Mink) with decreased activities of chloroplastic glutamine synthetase (GS) between 97 and 47% of the wild type and ferredoxin dependent glutamate synthase (Fd-GOGAT) down to 64% of the wild type have been used to study aspects of glyoxylate metabolism and the effect of glyoxylate on the activation state of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in vivo. In the leaf, the extractable activities of serine:glyoxylate aminotransferase decreased with a decrease in GS whereas activities of glutamate and alanine:glyoxylate aminotransferase increased, pointing to a re direction of amino donors from serine to glutamate and alanine. Under ambient conditions, the leaf contents of glutamate and alanine declined continuously with a decrease in GS, in parallel with the decrease in total amino acids. Glycine, serine and asparagine contents decreased with a decrease in GS to approximately 70% of the wild type, but increased again with a further decrease in GS. At high irradiances and at low CO₂ concentrations, glyoxylate contents exhibited a pronounced minimum between 60% and 80% GS. With a further decrease in GS, glyoxylate contents recovered and approached values similar to the wild type. The activation state of Rubisco showed a negative correlation with glyoxylate contents, indicating that a decrease in GS feeds back on the first step of carbon assimilation and photorespiration. The activation state of stromal fructose-1,6-bisphosphatase was unaffected by a decrease in GS or Fd-GOGAT, whereas the activation state of NADP dependent malate dehydrogenase changed in a complex manner. The CO₂photocompensation point, ^*, was appreciably increased in mutants with 47% GS. Mitochondrial respiration in the light (R_d) was reduced with a decrease in GS. Relative rates of CO₂ release into CO₂-free air between the wild type and the 47%-GS mutant correlated with determinations of ^*. These data are consistent with the view that when GS is decreased there is an increased oxidative decarboxylation of glyoxylate resulting from a decreased availability of amino donors for the transamination of glyoxylate to glycine, and that when GS activities are lower than 70% of the wild type an additional mechanism operates to reduce the photorespiratory loss of ammonia.Abbreviations AGAT nine:glyoxylate aminotransferase - FBPase fructose-1,6-bisphosphatase - Fd-GOGAT ferredoxin dependent glutamate synthase - GGAT glutamate:glyoxylate aminotransferase - GS glutamine synthetase - MDH malate dehydrogenase - PFD photon flux density - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - SGAT serine:glyoxylate aminotransferase This research was supported by the Biotechnology and Biological Sciences Research Council initiative on the Biochemistry of Metabolic Regulation in Plants (PG 50/555).     

Photorespiratory N donors,aminotransferase specificity and photosynthesis in a mutant of barley deficient in serine: glyoxylate aminotransferase activity

A mutant of Hordeum vulgare L. (LaPr 85/84) deficient in serine: glyoxylate aminotransferase (EC 2.6.1.45) activity has been isolated. The plant also lacks serine: pyruvate aminotransferase and asparagine: glyoxylate aminotransferase activities. Genetic analysis of the mutation strongly indicates that these three activities are all carried on the same enzyme protein. The mutant is incapable of normal rates of photosynthesis in air but can be maintained at 0.7% CO₂. The rate of photosynthesis cannot be restored by supplying hydroxypyruvate, glycerate, glutamate or ammonium sulphate through the xylem stream. This photorespiratory mutant demonstrates convincingly that photorespiration still occurs under conditions in which photosynthesis becomes insensitive to oxygen levels. Two major peaks and one minor peak of serine: glyoxylate aminotransferase activity can be separated in extracts of leaves of wild-type barley by diethylaminoethyl-sephacel chromatography. All three peaks are missing from the mutant, LaPr 85/84. The mutant showed the expected rate (50%) of ammonia release during photorespiration but produced CO₂ at twice the wild-type rate when it was fed [¹⁴C]glyoxylate. The large accumulation of serine detected in the mutant under photorespiratory conditions shows the importance of the enzyme activity in vivo. The effect of the mutation on transient changes in chlorophyll a fluorescence initiated by changing the atmospheric CO₂ concentration are presented and the role of the enzyme activity under nonphotorespiratory conditions is discussed.Abbreviations DEAE diethylaminoethyl - PFR photon fluence rate - SGAT serine:glyoxylate aminotransferase     

Asparagine and glutamine metabolism in Rhodopseudomonas acidophila

Rhodopseudomonas acidophila strain 7050 achieved balanced growth when provided with either asparagine or glutamine as nitrogen source. Under these growth conditions R. acidophila synthesized a mixed amidase which exhibited similar activity (223–422 nmol/min·mg protein) against either nitrogen source. Determination of the free intracellular amino acid pools show that deamidation of asparagine and glutamine resulted in elevated levels of both aspartate and glutamate. Cell-free extracts of R. acidophila showed significant aminotransferase activity, particulary glutamine-oxaloacetate aminotransferase (89.7–209.3 nmol/min·mg protein), glycine oxaloacetate aminotransferase (135–227 nmol/min ·mg protein), alanine glyoxylate aminotransferase (66.3–163.2 nmol/min·mg protein) and serineglyoxylate aminotransferase (57.1–68.4 nmol/min ·mg protein). Short term labelling experiments using ¹⁴C-glyoxylate show that glycine plays an important role in amino nitrogen transfer in R. acidophila and that the enzymes for the metabolism of glyoxylate via glycine, serine and hydroxypyruvate were present in cell-free extracts. These data confirm that R. acidophila can satisfy all its' nitrogen requirements by transamination.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase - MSO methionine sulfoximine - GOT glutamate—oxaloacetate aminotransferase - GPT glutamate-pyruvate aminotransferase - AGAT alanineglyoxylate aminotransferase - GOAT glycine-oxaloacetate aminotransferase - GOGAT glycine-2-oxoglutarate aminotransferase - AOAT alanine-oxaloacetate aminotransferase - SGAT serineglyoxylate aminotransferase - INH isonicotinylhydrazide     

A Mutant of Nicotiana sylvestris Lacking Serine:Glyoxylate Aminotransferase: Substrate Specificity of the Enzyme and Fate of [2-C]Glycolate in Plants with Genetically Altered Enzyme Levels

The photorespiratory mutant of Nicotiana sylvestris, NS 349, lacking serine:glyoxylate aminotransferase (SGAT) grows in 1% CO₂ but not in normal air (NA McHale, EA Havir, I Zelitch 1988 Theor Appl Genet. In press). Alanine:hydroxypyruvate and asparagine:hydroxypyruvate aminotransferase activities were also lacking in the mutant, and plants heterozygous with respect to SGAT which grow in normal air had 50% of the activities present in homozygous plants. Therefore, all these activities are associated with the same enzyme. On feeding [2-¹⁴C]glycolate to leaf discs in the light, NS 349 showed reduced incorporation of radioactivity into the neutral and organic acid fractions and increased incorporation into the amino acid fraction, principally into serine. The effect of reducing SGAT by 50% in heterozygous plants produced little change in the metabolism of [2-¹⁴C]glycolate, showing there is a large excess of this enzyme in wild-type plants.     

Overexpression of the Arabidopsis photorespiratory pathway gene, serine: glyoxylate aminotransferase (AtAGT1), leads to salt stress tolerance in transgenic duckweed (Lemna minor)

Salt stress has attracted increasing attention due to its toxic ability to restrict plant growth, and the photorespiration pathway has been shown to develop improved plant tolerance to abiotic stress. In this study, an Arabidopsis photorespiratory pathway gene serine: glyoxylate aminotransferase (SGAT), named as AtAGT1, was successfully overexpressed in duckweed (Lemna minor) to investigate the salinity defense capability in three transgenic overexpressed (OE) lines. Increased SGAT activity and decreased endogenous serine levels in these transgenic plant lines under salt stress resulted in enhanced protection against root abscission, higher maximum quantum yield of photosystem II (Fv/Fm), increased defense from cell damage as a result of improved cell membrane integrity, a decrease of reactive oxygen species (ROS) accumulation, and a strengthened antioxidant system. The salt tolerance in these transgenic OE lines indicates that the improvement of photorespiration stimulated the antioxidant system to scavenge ROS. The change of serine level also suggests the role of serine during salt stress. This transgenic engineering in duckweed not only introduced salt tolerance to this aquatic plant but also reveals a significant role of photorespiration during salinity stress.     

A mutant of Nicotiana sylvestris deficient in serine glyoxylate aminotransferase activity

Summary A photorespiration mutant of Nicotiana sylvestris lacking serine: glyoxylate aminotransferase activity was isolated in the M₂ generation following EMS mutagenesis. Mutants showing chlorosis in air and normal growth in 1% CO₂ were fed [¹⁴C]-2-glycolate to examine the distribution of ¹⁴C among photorespiratory intermediates. Mutant strain NS 349 displayed a 9-fold increase in serine accumulation relative to wild-type controls. Enzyme assays revealed an absence of serine: glyoxylate aminotransferase (SGAT) activity in NS 349, whereas other peroxisomal enzymes were recovered at normal levels. Heterozygous siblings of NS 349 segregating air-sensitive M₃ progeny in a 31 ratio were shown to contain one half the normal level of SGAT activity, indicating that air sensitivity in NS 349 results from a single nuclear recessive mutation eliminating SGAT activity. Since toxicity of the mutation depends on photorespiratory activity, callus cultures of the mutant were initiated and maintained under conditions suppressing the formation of functional plastids. Plantlets regenerated from mutant callus were shown to retain the SGAT deficiency and conditional lethality in air. The utility of photorespiration mutants of tobacco as vehicles for genetic manipulation of ribulose bisphosphate carboxylase/oxygenase at the somatic cell level is discussed.     

Properties of serine：glyoxylate aminotransferase purified from Arabidopsis thaliana leaves

The photorespiratory enzyme L-serine：glyoxylate amino- transferase （SGAT; EC 2.6.1.45） was purified from Arabidopsis thaliana leaves. The f＇mal enzyme was approximately 80 % pure as revealed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis with silver staining. The identity of the enzyme was confirmed by LC/MS/MS analysis. The molecular mass estimated by gel filtration chromato- graphy on Sephadex G-150 under non-denaturing conditions, mass spectrometry （matrix-assisted laser desorption/ ionization/time of flight technique） and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 82.4 kDa, 42.0 kDa, and 39.8 kDa, respectively, indicating dimer as the active form. The optimum pH value was 9.2. The enzyme activity was inhibited by aminooxyacetate and β-chloro-L-alanine both compounds reacting with the carbonyl group of pyridoxal phosphate. The enzyme＇s transaminating activity with L-alanine and glyoxylate as substrates was approximately 55 % of that observed with L-serine and glyoxylate. The lower Kmvalue （1.25 mM） for L-alanine, compared with that of other plant SGATs, and the kcat/Km（Ala） ratio being approxi- mately 2-fold higher than kcat/Km（Ser） suggested that, during photorespiration, Ala and Ser are used by Arabidopsis SGAT with equal efficiency as amino group donors for glyoxylate. The equilibrium constant （Keq）, derived from the Haldane relation, for the transamination reaction between L-serine and glyoxylate with the formation of hydroxypyruvate and glycine was 79.1, strongly favoring glycine synthesis. However, it was accompanied by a low Km value of 2.83 mM for glycine. A comparison of some kinetic properties of the studied enzymes with the recombinant Arabidopsis SGATs previously obtained revealed substantial differences. The ratio of the velocity of the transamination reaction with L-alanine and glyoxylate as substrates versus that with L-serine and glyoxylate was 1：1.8 for the native enzyme, whereas it was 1：7 for the recombinant SGAT. Native SGAT showed a much lower Km value for L-alanine compared to the recombinant enzyme.     

Serine: glyoxylate, alanine:glyoxylate, and glutamate:glyoxylate aminotransferase reactions in peroxisomes from spinach leaves

Two different aminotransferases, that have glyoxylate as the amino acceptor, have specific activities of 1 to 2 mumol . min-1 . mg of protein-1 in the isolated peroxisomal fraction from spinach leaves. Their properties were evaluated after separation on a hydroxylapatite column. Both enzymes had a Km for glyoxylate of 0.15 mM and an amino acid Km of 2 to 3 mM. Reactions proceeded by a Ping Pong Bi Bi mechanism. Serine:glyoxylate aminotransferase was relatively specific for both substrates and could only be slightly reversed with 100 mM glycine, although the Ki of glycine was 33 mM. The glutamate:glyoxylate amino-transferase protein was equally active in catalyzing an alanine:glyoxylate aminotransferase reaction, but the reverse reactions with 100 mM glycine were hardly measureable, although the Ki (glycine) was 8.7 mM. Protection against hydroxylamine inhibition from reaction with pyridoxal phosphate was used to investigate the specificity of amino acid binding. Substrate amino acids protected at about the same concentration as their Km, while glycine protected at its Ki concentration. Thus, the nearly irreversible catalysis with glycine is not due to a failure to bind glycine. The significance of a peroxisomal alanine:glyoxylate aminotransferase activity has not been incorporated into schemes for the oxidative photosynthetic carbon cycle.     

Glyoxylate transamination in intact leaf peroxisomes

Intact spinach (Spinacia oleracea L.) leaf peroxisomes were supplied with glycolate and one to three of the amino acids serine, glutamate, and alanine, and the amount of the respective α-keto acids formed in glyoxylate transamination was assayed. At 1 millimolar glycolate and 1 millimolar each of the three amino acids in combination, the transamination reaction reached saturation; reduction of either glycolate or amino acid concentration decreased the activity. The relative serine, glutamate, and alanine transamination at equal amino acid concentrations was roughly 40, 30, and 30%, respectively. The three amino acids exhibited mutual inhibition to one another in transamination due to the competition for the supply of glyoxylate. In addition to this competition for glyoxylate, competitive inhibition at the active site of enzymes occurred between glutamate and alanine, but not between serine and glutamate or alanine. Alteration of the relative concentrations of the three amino acids changed their relative transamination. Similar work was performed with intact oat (Avena sativa L.) leaf peroxisomes. At 1 millimolar of each of the three amino acids in combination, the relative serine, glutamate, and alanine transamination was roughly 60, 23, and 17%, respectively. Similarly, alteration of the relative concentration of the three amino acids changed their relative transamination. The contents of the three amino acids in leaf extracts were analyzed, and the relative contribution of the three amino acids in glycine production in photorespiration was assessed and discussed.     

Role of asparagine in the photorespiratory nitrogen metabolism of pea leaves

In pea leaves, much of the metabolism of imported asparagine is by transamination. This activity was previously shown to be localized in the peroxisomes, suggesting a possible connection between asparagine and photorespiratory nitrogen metabolism. This was investigated by examination of the transfer of ¹⁵N from the amino group of asparagine, supplied via the transpiration stream, in fully expanded pea leaves. Label was transferred to aspartate, glutamate, alanine, glycine, serine, ammonia, and glutamine (amide group). Under low oxygen (1.8%), or in the presence of α-hydroxy-2-pyridine methanesulfonic acid (an inhibitor of glycolate oxidase, a step in the photorespiratory formation of glyoxylate), there was a substantial (60-80%) decrease in transfer of label to glycine, serine, ammonia, and glutamine. Addition of isonicotinyl hydrazide (an inhibitor of formation of serine from glycine) caused a 70% decrease in transfer of asparagine amino nitrogen to serine, ammonia, and glutamine, while a 4-fold increase in labeling of glycine was observed. The results demonstrate the involvement of asparagine in photorespiration, and show that photorespiratory nitrogen metabolism is not a closed cyclic process.     

Reactivity of glyoxylate with hydrogen perioxide and simulation of the glycolate pathway of C3 plants and Euglena

The nonenzymatic reaction of glyoxylate and H₂O₂ was measured under physiological conditions of the pH and concentrations of reactants. The reaction of glyoxylate and H₂O₂ was secondorder, with a rate constant of 2.27 l mol^-1 s^-1 at pH 8.0 and 25° C. The rate constant increased by 4.4 times in the presence of Zn²⁺ and doubled at 35°C. We propose a mechanism for the reaction between glyoxylate and H₂O₂. From a comparison of the rates of H₂O₂ decomposition by catalase and the reaction with glyoxylate, we conclude that H₂O₂ produced during glycolate oxidation in peroxisomes is decomposed by catalase but not by the reaction with glyoxylate, and that photorespiratory CO₂ originates from glycine, but not from glyoxylate, in C₃ plants. Simulation using the above rate constant and reported kinetic parameters leads to the same conclusion, and also makes it clear that alanine is a satisfactory amino donor in the conversion of glyoxylate to glycine. Some serine might be decomposed to give glycine and methylene-tetrahydrofolate; the latter is ultimately oxidized to CO₂. In the simulation of the glycolate pathway of Euglena, the rate constant was high enough to ensure the decarboxylation of glyoxylate by H₂O₂ to produce photorespiratory CO₂ during the glycolate metabolism of this organism.Abbreviations Chl chlorophyll - GGT glutamate: glyoxylate aminotransferase (EC 2.6.1.4) - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - SGT serine: glyoxylate aminotransferase (EC 2.6.1.45) This is the ninth in a series on the metabolism of glycolate in Euglena gracilis. The eighth is Yokota et al. (1982)     

Biochemical characterization and kinetic properties of alanine aminotransferase homologues partially purified from wheat (Triticum aestivum L.)

Four homologues of alanine aminotransferase have been isolated from shoots of wheat seedlings and purified by saline precipitation, gel filtration, preparative electrophoresis and anion exchange chromatography on Protein-Pak Q 8HR column attached to HPLC. Alanine aminotransferase 1 (AlaAT1) and 2 (AlaAT2) were purified 303- and 452-fold, respectively, whereas l-glutamate: glyoxylate aminotransferase 1 (GGAT1) and 2 (GGAT2) were purified 485- and 440-fold, respectively. Consistent inhibition of AlaAT (EC 2.6.1.2) and GGAT (EC 2.6.1.4) activities by p-hydroxymercuribenzoate points on participation of cysteine residues in the enzyme activity. The molecular weight of AlaAT1 and AlaAT2 was estimated to be 65 kDa and both of them are monomers in native state. Nonsignificant differences between K_m using alanine as substrate and catalytic efficiency (k_cat/K_m) for l-alanine in reaction with 2-oxoglutarate indicate comparable kinetic constants for AlaAT1 and AlaAT2. Similar kinetic constants for l-alanine in reaction with 2-oxoglutarate and for l-glutamate in reaction with pyruvate for all four homologues suggest equally efficient reaction in both forward and reverse directions. GGAT1 and GGAT2 were able to catalyze transamination between l-glutamate and glyoxylate, l-alanine and glyoxylate and reverse reactions between glycine and 2-oxoglutarate or pyruvate. Both GGATs also consisted of a single subunit with molecular weight of about 50 kDa. The estimated K_m for GGAT1 (3.22 M) and GGAT2 (1.27 M) using l-glutamate as substrate was lower in transamination with glyoxylate than with pyruvate (9.52 and 9.09 mM, respectively). Moreover, distinctively higher values of catalytic efficiency for l-glutamate in reaction with glyoxylate than for l-glutamate in reaction with pyruvate confirm involvement of these homologues into photorespiratory metabolism.     

Analysis of glutamate homeostasis by overexpression of Fd-GOGAT gene in Arabidopsis thaliana

Glutamate plays a central role in nitrogen flow and serves as a nitrogen donor for the production of amino acids. In plants, some amino acids work as buffers: during photorespiration, ammonium derived from the conversion of glycine to serine is promptly reassimilated into glutamate by the glutamine synthetase (GS-2)/ferredoxin-dependent glutamate synthase (Fd-GOGAT) cycle. The glutamate concentration is relatively stable compared with those of other amino acids under environmental changes. The few studies dealing with glutamate homeostasis have but all used knockouts or mutants of these enzymes. Here, we generated Fd-GOGAT (GLU1)-overexpressing Arabidopsis plants to analyze changes in the amino acid pool caused by glutamate overproduction under different ammonium conditions controlled by CO₂ concentration, light intensity and nitrate concentration. Under photorespiratory conditions with sufficient ammonium supply, aspartate increased and glutamine and glycine decreased, but glutamate barely changed. Under non-photorespiratory conditions, however, glutamate and most other amino acids increased. These results suggest that the synthesized glutamate is promptly converted into other amino acids, especially aspartate. In addition, ammonium supply by photorespiration does not limit glutamate biosynthesis, but glutamine and glycine are important. This study will contribute to the understanding of glutamate homeostasis in plants.