Serine-glyoxylate Aminotransferase Research Articles

The Role of Serine-Glyoxylate Aminotransferase and Malyl-CoA Lyase in the Metabolism of Methylococcus capsulatus Bath.

The genome of aerobic methanotroph Methylococcus capsulatus Bath possesses genes of three biochemical pathways of C1-carbon assimilation: the ribulose monophosphate cycle, the Calvin-Benson-Bassham cycle, and the partial serine cycle. Numerous studies have demonstrated that during methanotrophic growth cells of Methylococcus capsulatus Bath express key enzymes of these routes. In this study, the role of the serine cycle key enzymes, serine-glyoxylate aminotransferase (Sga) and malyl-CoA lyase (Mcl) in metabolism of Methylococcus capsulatus Bath was investigated by gene inactivation. The Δsga mutant obtained by double homologous recombination showed a prolonged lag phase, and after the lag period, the growth rate became similar to that of the wild type strain. The elevated intracellular levels of glutamate, serine, glycine, alanine, methionine, leucine, and succinate suggested significant metabolic changes in the mutant cells. Deletion of the mcl gene resulted in very poor growth and glycine only partially improved growth of the mutant strain. Cells of Δmcl mutant possess lower content of histidine, but enhanced level of alanine, leucine, and lysine than those of the wild type strain. Our data imply the importance of the serine cycle enzymes in metabolism of the methanotroph as well as relationships of the three C1 assimilation pathways in the gammaproteobacterial methanotrophs.

Transcriptomic analysis of bakanae disease resistant and susceptible rice genotypes in response to infection by Fusarium fujikuroi.

Fusarium fujikuroi causing bakanae is one of the most significant pathogens of rice and much responsible for yield losses thereby emerging as a major risk to food security. In the present study transcriptomic analysis was conducted between two contrasting resistant (C101A51) and susceptible (Rasi) genotypes of rice with the combinations of C101A51 control (CC) vs. C101A51 inoculated (CI); Rasi control (RC) vs. Rasi inoculated (RI) and C101A51 inoculated (CI) vs. Rasi inoculated (RI). In CC vs. CI commonly expressed genes were 12,764. Out of them 567 (4%) were significantly upregulated and 1399 (9%) genes were downregulated. For the RC vs. RI 14, 333 (79%) genes were commonly expressed. For CI vs. RI 13,662 (72%) genes were commonly expressed. Genes related to cysteine proteinase inhibitor 10, disease resistance protein TAO1-like, oleosin 16kDa-like, pathogenesis-related protein (PR1), (PR4), BTB/POZ and MATH domain-containing protein 5-like, alpha-amylase isozyme were upregulated in resistant genotype C101A51. Whereas, genes related to GDSL esterase/lipase, serine glyoxylate aminotransferase, CASP-like protein 2C1, WAT1-related protein, Cytoplasmic linker associated proteins, xyloglucan endotransglucosylase/hydrolase protein and β-D xylosidase 7 were upregulated in susceptible genotype Rasi. Gene ontology analysis showed functions related to defence response (GO:0006952), regulation of plant hypersensitive type response (GO:0010363), Potassium ion transmembrane activity (GO:0015079), chloroplast (GO:0009507), response to wounding (GO:0009611), xylan biosynthetic process (GO:0045492) were upregulated in resistant genotype C101A51 under inoculated conditions. Real time PCR based validation of the selected DEGs showed that the qRT-PCR was consistent with the RNA-Seq results. This is the first transcriptomic study against bakanae disease of rice in Indian genotypes. Further, functional studies on identified genes and their utilization through different methodology will be helpful for the development of bakanae disease management strategies.

Methylotrophic bacterium-based molecular sensor for the detection of low concentrations of methanol

Methylotrophic bacterium Methylorubrum extorquens is a promising microorganism for the production of value-added compounds from methanol. This study focused on the development of a single-cell level biosensor system that detects methanol by using the intrinsic regulatory machinery which responds to the presence of methanol in this bacterium. A green fluorescent protein (GFP) gene located downstream of the promoter region of the serine glyoxylate aminotransferase gene (Psga) or the methanol dehydrogenase subunit 1 precursor gene (PmxaF) was inserted into the chromosome of M.extorquens wild-type strain AM1. The expression of GFP upon methanol exposure was measured by spectrofluorometer and fluorescence-activated cell sorting (FACS). The strain harboring Psga-gfp emitted fluorescence only when methanol was supplied to the culture medium, while the other strain harboring PmxaF-gfp showed high basal fluorescence even in the absence of methanol. The fluorescence intensity of the Psga-gfp strain depended on a methanol concentration higher than 25μM, and the sensitivity and dose-dependency of this strain were much higher than previous systems using Escherichia coli. The methanol-sensing properties of the engineered M.extorquens strain were comparable to those of a methylotrophic yeast-based biosensor, suggesting the usefulness of methylotrophic microorganisms as platforms for single-cell sensing of C1 compounds. The constructed methanol sensor strain, coupled with flow cytometry techniques, provides a high-throughput and highly sensitive screening method for the selection of functional methanol-producing enzymes.

Proteomic analysis of hydrogen production in Chlorella pyrenoidosa under nitrogen deprivation

Previously, Chlorella pyrenoidosa strain IOAC707S was found to produce hydrogen under nitrogen deprivation in seawater medium, without dark incubation, N2 flushing or addition of uncoupler, which made it a candidate for clean and renewable energy production. To understand the molecular mechanism underlying hydrogen production by C. pyrenoidosa under nitrogen deprivation, iTRAQ-based proteomic analysis was conducted to investigate the differences in protein abundance under nitrogen deprivation (L1-N) compared with the control (CK). In total, 119 KEGG pathways were mapped for 2383 proteins, among which, the expression levels of most proteins involved in chlorophyll synthesis were down-regulated, while those involved in chlorophyll degradation were up-regulated in the L1-N culture; the expression level of serine glyoxylate aminotransferase involved in photorespiration was up-regulated 1.25 fold in the L1-N culture at 44 h, and the expression levels of most proteins involved in mitochondrial respiration were up-regulated in the L1-N culture. These results indicated a decrease of chlorophyll and an increase of respiration, which favored the establishment of anaerobiosis and hydrogen production.

Chronic effects of microcystin-LR at environmental relevant concentrations on photosynthesis of Typha angustifolia Linn.

Understanding the growth and development of aquatic plants in eutrophic water is of great significance for the selection of potential candidate plant for use in the phytoremediation of eutrophic aquatic ecosystems. The present study aimed to investigate the chronic effects of microcystin-LR (MC-LR) on photosynthesis in the leaves of Typha angustifolia Linn. Photosynthetic activity was stimulated in the leaves following exposure to 4.6 μg L-1 MC-LR for six weeks based on the enhancement of Ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) activity and net photosynthetic rate (PN). However, PN decreased significantly after exposure to 49.1 or 98.3 μg L-1 MC-LR, via non-stomatal limitation by reducing the chlorophyll a and b contents and Rubisco activity. In addition, glycolate oxidase (GO) and serine:glyoxylate aminotransferase (SGAT) activities decreased significantly, indicating that the photorespiration pathway was affected adversely. The intercellular carbon dioxide (Ci) concentration decreased significantly following exposure to 98.3 μg L-1 MC-LR, accompanied with decreases in PN and stomatal conductivity (gs), indicating that stomatal limitation on the photosynthesis system in T. angustifolia L. was observed after exposure to 98.3 μg L-1 MC-LR. Under long-term exposure to MC-LR (49.1-98.3 μg L-1), oxidation stress was severe in the aquatic plant, and non-stomatal limitation or stomatal limitation effects on the photosynthesis system were obvious, resulting in decreases in photosynthesis rates.

Serine-glyoxylate aminotranferases from methanotrophs using different C1-assimilation pathways.

The indicator enzyme of the serine pathway of assimilation of reduced C1 compounds, serine-glyoxylate aminotransferase (Sga), has been purified from three methane-oxidizing bacteria, Methylomicrobium alcaliphilum 20Z, Methylosinus trichosporium OB3b and Methylococcus capsulatus Bath. The native enzymes were shown to be dimeric (80kDa, strain 20Z), tetrameric (~ 170kDa, strain OB3b) or trimeric (~ 120kDa, strain Bath). Sga from the three methanotrophs catalyse the pyridoxal phosphate-dependent transfer of an amino group from serine to glyoxylate and pyruvate; the enzymes from strains 20Z and Bath also transfer an amino group from serine to α-ketoglutarate and from alanine to glyoxylate. No other significant differences between the Sga from the three methanotrophs were found. The three methanotrophic Sga have their highest catalytic efficiencies in the reaction between glyoxylate and serine, which is in agreement with their function to provide circulation of the serine assimilation pathway.The disruption of the sga gene in Mm. alcaliphilum resulted in retardation of growth rate of the mutant cells and in a prolonged lag-phase after passaging from methane to methanol. In addition, the growth of the mutant strain is accompanied by formaldehyde accumulation in the culture liquid. Hence, Sga is important in the serine cycle of type I methanotrophs and this pathway could be related to the removal of excess formaldehyde and/or energy regulation.

Paracoccus simplex sp. nov., a New Methylamine-Utilizing Facultative Methylotroph

A facultative methylotroph, strain F5Т, which uses methylamine and a broad spectrum of polycarbon substrates as carbon and energy sources, was isolated from silt of a freshwater lake in the southern Moscow region. The cells were gram-negative, coccoid, non-spore-forming, nonmotile, colorless, reproducing by binary fission and possessing a capsule. The organism was mesophilic, neutrophilic, not halophilic, oxidase- and catalase-positive, and capable of nitrate reduction to nitrite. Methylamine was oxidized by amine dehydrogenase and via the icl- serine pathway of С1 metabolism, as was indicated by activities of hydroxypyruvate reductase and serine-glyoxylate aminotransferase and by the absence of hexulosephosphate synthase and ribulose bisphosphate carboxylase. Predominant fatty acids were C18:1ω7c (72.3%) and C16:0 (11.6%). Phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, and phosphatidylethanolamine were the dominant phospholipids. The G + C DNA content was 65.8 mol % (Tm). Q10 was the dominant ubiquinone. Strain F5T exhibited high similarity of the 16S rRNA gene sequences with Paracoccus strains: P. aminovorans JCM7685T = VKM B-2140Т (98.0%), P. huijuniae FLN-7Т (97.9%), and P. limosus NB88T (97.5%). However, the level of DNA-DNA relatedness between the strains F5Т and P. aminovoransТ was only 21 ± 3%. Based on the data obtained, strain F5Т was identified as a new Paracoccus species with proposed name Paracoccus simplex sp. nov. (= VKM B-3226T = CCUG 71989T).

Genes for asparagine metabolism in Lotus japonicus: differential expression and interconnection with photorespiration

BackgroundAsparagine is a very important nitrogen transport and storage compound in plants due to its high nitrogen/carbon ratio and stability. Asparagine intracellular concentration depends on a balance between asparagine biosynthesis and degradation. The main enzymes involved in asparagine metabolism are asparagine synthetase (ASN), asparaginase (NSE) and serine-glyoxylate aminotransferase (SGAT). The study of the genes encoding for these enzymes in the model legume Lotus japonicus is of particular interest since it has been proposed that asparagine is the principal molecule used to transport reduced nitrogen within the plant in most temperate legumes.ResultsA differential expression of genes encoding for several enzymes involved in asparagine metabolism was detected in L. japonicus. ASN is encoded by three genes, LjASN1 was the most highly expressed in mature leaves while LjASN2 expression was negligible and LjASN3 showed a low expression in this organ, suggesting that LjASN1 is the main gene responsible for asparagine synthesis in mature leaves. In young leaves, LjASN3 was the only ASN gene expressed although at low levels, while all the three genes encoding for NSE were highly expressed, especially LjNSE1. In nodules, LjASN2 and LjNSE2 were the most highly expressed genes, suggesting an important role for these genes in this organ. Several lines of evidence support the connection between asparagine metabolic genes and photorespiration in L. japonicus: a) a mutant plant deficient in LjNSE1 showed a dramatic decrease in the expression of the two genes encoding for SGAT; b) expression of the genes involved in asparagine metabolism is altered in a photorespiratory mutant lacking plastidic glutamine synthetase; c) a clustering analysis indicated a similar pattern of expression among several genes involved in photorespiratory and asparagine metabolism, indicating a clear link between LjASN1 and LjSGAT genes and photorespiration.ConclusionsThe results obtained in this paper indicate the existence of a differential expression of asparagine metabolic genes in L. japonicus and point out the crucial relevance of particular genes in different organs. Moreover, the data presented establish clear links between asparagine and photorespiratory metabolic genes in this plant.

Tomato photorespiratory glycolate-oxidase-derived H2 O2 production contributes to basal defence against Pseudomonas syringae.

Despite being essential for C3 plants, photorespiration is believed to cause a significant crop yield loss even under future climates. However, how photorespiration affects plant basal defence still remains largely unknown. Here, we studied the involvement of photorespiration in tomato-Pseudomonas syringae pv. tomato DC3000 interaction focusing on three photorespiratory genes. Inoculation with P.syringae increased photorespiration rate (Pr) and expression of glycolate oxidase (GOX2), serine glyoxylate aminotransferase (SGT) and serine hydroxyl methyltransferase (SHMT1); however, inhibition of photorespiration by isonicotinic acid hydrazide decreased tomato basal defence against P.syringae. Furthermore, silencing of GOX2, SGT or SHMT1 genes in tomato decreased Pr but increased susceptibility to P.syringae, whereas transient overexpression of GOX2, SGT or SHMT1 in tobacco increased basal defence. Further study revealed that salicylic acid (SA) signalling is involved in GOX2-mediated, SGT-mediated and SHMT1-mediated defence. Moreover, H2 O2 pretreatment remarkably alleviated the GOX2 silencing-induced depression in basal defence and SA signalling, whereas it had no effect on that of SGT-silenced and SHMT1-silenced plants. Taken together, these results suggest that H2 O2 is critical for GOX2-modulated but not SGT-modulated or SHMT1-modulated SA signalling and subsequent basal defence against P.syringae. This work deepens the understanding of photorespiration-involved defence responses to bacterial attack in plants.

High serine:glyoxylate aminotransferase activity lowers leaf daytime serine levels, inducing the phosphoserine pathway in Arabidopsis.

Serine:glyoxylate aminotransferase (SGAT) converts glyoxylate and serine to glycine and hydroxypyruvate during photorespiration. Besides this, SGAT operates with several other substrates including asparagine. The impact of this enzymatic promiscuity on plant metabolism, particularly photorespiration and serine biosynthesis, is poorly understood. We found that elevated SGAT activity causes surprisingly clear changes in metabolism and interferes with photosynthetic CO2 uptake and biomass accumulation of Arabidopsis. The faster serine turnover during photorespiration progressively lowers day-time leaf serine contents and in turn induces the phosphoserine pathway. Transcriptional upregulation of this additional route of serine biosynthesis occurs already during the day but particularly at night, efficiently counteracting night-time serine depletion. Additionally, higher SGAT activity results in an increased use of asparagine as the external donor of amino groups to the photorespiratory pathway but does not alter leaf asparagine content at night. These results suggest leaf SGAT activity needs to be dynamically adjusted to ensure (i) variable flux through the photorespiratory pathway at a minimal consumption of asparagine and (ii) adequate serine levels for other cellular metabolism.

Distinct photorespiratory reactions are preferentially catalyzed by glutamate:glyoxylate and serine:glyoxylate aminotransferases in rice

The metabolic function of glutamate:glyoxylate aminotransferase (GGAT) and serine:glyoxylate aminotransferase (SGAT) for photorespiration is still not exactly understood so far though it is mostly held that both enzymes may work in parallel in the reaction of glyoxylate to glycine during photorespiration of plants. Here, for the first time, we define the genes encoding GGAT and SGAT and report their biochemical and enzymatic properties in rice plants, in contrast to those from other plant species. Noticeably, GGAT exhibited approximately 18 fold higher catalytic efficiency (Kcat/Km) with glyoxylate and glutamate than SGAT with glyoxylate and serine, and additionally, rice leaves usually contain 3–4times higher abundance of glutamate relative to serine, implicating that GGAT may preferentially utilize glyoxylate to form glycine over SGAT. When SGAT or GGAT activity was regulated by gene transformation or nitrogen deficiency, respectively, it was observed that the glycine content was positively related to GGAT activities, while both serine and glycine contents were negatively related to SGAT activities. The results suggest that GGAT preferentially catalyzes the conversion of glyoxylate into glycine while SGAT is mainly responsible for the transamination reaction of serine to hydroxypyruvate in the photorespiratory pathway of rice.

ROS production is associated with glycolate metabolism in Chlamydomonas reinhardtii (Chlorophyceae) under salt stress

:The photorespiration metabolism in unicellular algae is different from that in higher plants because of the absence of peroxisomes, and conversion of glycolate to glyoxylate is catalyzed by glycolate dehydrogenase without the production of reactive oxygen species (ROS). It has been reported that the glycolate–quinone oxidoreductase system (GQOS) also plays a role in the formation of glyoxylate from glycolate that is coupled to photosynthetic electron transport. In this study, we showed that production of ROS was associated with the oxidation of glycolate in chloroplasts of Chlamydomonas reinhardtii cells and that both ROS accumulation and photorespiration were promoted by NaCl stress. Salicylhydroxamic acid (SHAM) and methoxylamine (MOA) are inhibitors that block effectively the GQOS oxidation of glycolate and glycine decarboxylase complex, respectively. Our data showed that in the dark, whether or not the cells were treated with inhibitor, no remarkable difference was found in ROS levels under the same NaCl stress. In the light, however, the ROS level in the cells treated with SHAM or MOA was significantly lower than that in nontreated cells, with SHAM being more effective. The increase in serine:glyoxylate aminotransferase (SGAT) activity and glycine/serine (Gly/Ser) ratio were 8.3% and 30.8% under 50 mM, and reached 34.4% and 1.5-fold under 100 mM NaCl stress.

Two Hydroxypyruvate Reductases Encoded by OsHPR1 and OsHPR2 are Involved in Photorespiratory Metabolism in Rice

Mutations in the photorespiration pathway display a lethal phenotype in atmospheric air, which can be fully recovered by elevated CO2 . An exception is that mutants of peroxisomal hydroxypyruvate reductase (HPR1) do not have this phenotype, indicating the presence of cytosolic bypass in the photorespiration pathway. In this study, we constructed overexpression of the OsHPR1 gene and RNA interference plants of OsHPR1 and OsHPR2 genes in rice (Oryza sativa L. cv. Zhonghua 11). Results from reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and enzyme assays showed that HPR1 activity changed significantly in corresponding transgenic lines without any effect on HPR2 activity, which is the same for HPR2. However, metabolite analysis and the serine glyoxylate aminotransferase (SGAT) activity assay showed that the metabolite flux of photorespiration was disturbed in RNAi lines of both HPR genes. Furthermore, HPR1 and HPR2 proteins were located to the peroxisome and cytosol, respectively, by transient expression experiment. Double mutant hpr1 × hpr2 was generated by crossing individual mutant of hpr1 and hpr2. The phenotypes of all transgenic lines were determined in ambient air and CO2 -elevated air. The phenotype typical of photorespiration mutants was observed only where activity of both HPR1 and HPR2 were downregulated in the same line. These findings demonstrate that two hydroxypyruvate reductases encoded by OsHPR1 and OsHPR2 are involved in photorespiratory metabolism in rice.

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.

The photorespiratory pathway is involved in the defense response to powdery mildew infection in chestnut rose

Chestnut rose (Rosa roxburghii Tratt), a non-domesticated potential fruit crop, is susceptible to infection by powdery mildew. A small-scale customized macro-array was assembled with cDNA clones from a suppression subtractive hybridization library enriched for defense transcripts and used to screen for differentially expressed genes induced by powdery mildew infection. Three photorespiratory genes, encoding the small subunit of RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase), RuBisCO activase and serine-glyoxylate aminotransferase, were identified in this screen. Quantitative RT-PCR analysis of these three genes showed that they were significantly up-regulated 16 h after the host was inoculated with powdery mildew, and assays at the enzyme level confirmed high levels of enzymatic activity 24 h after infection. Of six phytohormones tested, salicylic acid (SA) noticeably induced their expression, and HPLC analysis showed that SA accumulated in the leaves after fungal infection.

Photorespiration Process and Nitrogen Metabolism in Lettuce Plants (Lactuca sativa L.): Induced Changes in Response to Iodine Biofortification

Iodine is vital to human health, and iodine biofortification programs help improve human intake through plant consumption. There is no research on whether iodine biofortification influences basic plant physiological processes. Because nitrogen (N) uptake, utilization, and accumulation are determining factors in crop yield, the aim of this work was to establish the effect of the application of different doses (20, 40, and 80 μM) and forms of iodine (iodate [IO3 −] vs. Iodide [I−]) on N metabolism and photorespiration. For this study we analyzed shoot biomass and the activities of nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AAT), glutamate dehydrogenase (GDH), glycolate oxidase (GO), glutamate:glyoxylate aminotransferase (GGAT), serine:glyoxylate aminotransferase (SGAT), hydroxypyruvate reductase (HR) and catalase (CAT), nitrate (NO3 −), ammonium (NH4 +), organic and total N, amino acids, proteins, serine (ser), malate, and α-ketoglutaric acid in edible lettuce leaves. Application of I− at doses of at least 40 μM reduced the foliar concentration of NO3 − with no decrease in biomass production, which may improve the nutritional quality of lettuce plants. In contrast, the application of 80 μM of I− is phytotoxic for lettuce plants, reducing the biomass, foliar concentration of organic N and NO3 −, and NR and GDH activities. HR activity is significantly inhibited with all doses of I−; the least inhibition was at 80 μM. This may involve a decrease in the incorporation of carbonated skeletons from photorespiration into the Calvin cycle, which may be partially associated with the biomass decrease. Finally, the application of IO3 − increases biomass production, stimulates NO3 − reduction and NH4 + incorporation (GS/GOGAT), and optimizes the photorespiratory process. Hence, this appears to be the most appropriate form of iodine from an agronomic standpoint.

Glyoxylate rather than ascorbate is an efficient precursor for oxalate biosynthesis in rice

Oxalate is widely distributed in the plant kingdom. While excess oxalate in food crops is detrimental to animal and human health, it may play various functional roles in plants, particularly for coping with environmental stresses. Understanding its biosynthetic mechanism in plants, therefore, becomes increasingly important both theoretically and practically. However, it is still a matter of debate as to what precursor and pathway are ultimately used for oxalate biosynthesis in plants. In this study, both physiological and molecular approaches were applied to address these questions. First, it was observed that when glycolate or glyoxylate was fed into detached leaves, both organic acids were equally effective in stimulating oxalate accumulation. In addition, the stimulation could be completely inhibited by cysteine, a glyoxylate scavenger that forms cysteine-glyoxylate adducts. To verify the role of glyoxylate further, various transgenic plants were generated, in which several genes involved in glyoxylate metabolism [i.e. SGAT (serine-glyoxylate aminotransferase), GGAT (glutamate-glyoxylate aminotransferase), HPR (hydroxypyruvate reductase), ICL (isocitrate lyase)], were transcriptionally regulated through RNAi or over-expression. Analyses on these transgenic plants consistently revealed that glyoxylate acted as an efficient precursor for oxalate biosynthesis in rice. Unexpectedly, it was found that oxalate accumulation was not correlated with photorespiration, even though this pathway is known to be a major source of glyoxylate. Further, when GLDH (L-galactono-1,4-lactone dehydrogenase), a key enzyme gene for ascorbate biosynthesis, was down-regulated, the oxalate abundance remained constant, despite ascorbate having been largely reduced as expected in these transgenic plants. Taken together, our results strongly suggest that glyoxylate rather than ascorbate is an efficient precursor for oxalate biosynthesis, and that oxalate accumulation and regulation do not necessarily depend on photorespiration, possibly due to the occurrence of the anaplerotic reaction that may compensate for glyoxylate formation in rice.

Detection of a gem-diamine and a stable quinonoid intermediate in the reaction catalyzed by serine–glyoxylate aminotransferase from Hyphomicrobium methylovorum

BackgroundThe enzyme l-serine–glyoxylate aminotransferase (SGAT) from Hyphomicrobium methylovorum is a PLP-containing enzyme that catalyzes the conversion of l-serine and glyoxylate to hydroxypyruvate and glycine. The cloned enzyme expressed in Escherichia coli is isolated as a mixture of the E:PLP and E:PMP forms. The PLP form of the enzyme has a maximum absorbance at 413 nm. MethodsUv–visible spectra of SGAT were obtained using an HP-8453 diode array spectrophotometer in the absence and presence of substrates and substrate analogs. Pre-steady state kinetic studies were carried out using an OLIS rapid scanning spectrophotometer in the rapid scanning mode. ResultsIncubation of the enzyme with a saturating concentration D-serine leads to a shift in the 413 nm peak to 421 nm that is ascribed to the external aldimine. The reverse stereochemistry of D-serine does not allow for abstraction of the Cα proton by the ε-amine of the active site lysine residue leading to an abortive external aldimine intermediate. Pre-steady state studies pushing SGAT against d-serine leads to a rapid decrease in the 413 nm peak and an increase at ∼330 nm with an associated rate constant of 47 s−1 at pH 7.6. This is followed by a slower decrease (0.26 s−1) at 330 nm and an increase and shift of the 413 nm peak to 421 nm. The intermediate species that absorbs at ∼330 nm is attributed to the gem-diamine intermediate. The rate of the fast phase increases with pH and increase in rate is likely due to the deprotonation of an enzymatic group that accepts a proton from the α-amine of d-serine. In the presence of hydroxypyruvate and ammonia the enzyme spectra display an increase in absorbance at 521 nm that occurs on the order of minutes. The shape and position of the 521 nm species is consistent with a quinonoid intermediate. General significanceThe data suggest a non-enzymatic reaction between hydroxypyruvate and ammonia to form an imine which will be in equilibrium with the enamine. A mechanism is proposed by which the enamine reacts with the PLP form of SGAT to generate the stable highly conjugated quinonoid intermediate.

Shewanella oneidensis MR-1 Fluxome under Various Oxygen Conditions

The central metabolic fluxes of Shewanella oneidensis MR-1 were examined under carbon-limited (aerobic) and oxygen-limited (microaerobic) chemostat conditions, using 13C-labeled lactate as the sole carbon source. The carbon labeling patterns of key amino acids in biomass were probed using both gas chromatography-mass spectrometry (GC-MS) and 13C nuclear magnetic resonance (NMR). Based on the genome annotation, a metabolic pathway model was constructed to quantify the central metabolic flux distributions. The model showed that the tricarboxylic acid (TCA) cycle is the major carbon metabolism route under both conditions. The Entner-Doudoroff and pentose phosphate pathways were utilized primarily for biomass synthesis (with a flux below 5% of the lactate uptake rate). The anaplerotic reactions (pyruvate to malate and oxaloacetate to phosphoenolpyruvate) and the glyoxylate shunt were active. Under carbon-limited conditions, a substantial amount (9% of the lactate uptake rate) of carbon entered the highly reversible serine metabolic pathway. Under microaerobic conditions, fluxes through the TCA cycle decreased and acetate production increased compared to what was found for carbon-limited conditions, and the flux from glyoxylate to glycine (serine-glyoxylate aminotransferase) became measurable. Although the flux distributions under aerobic, microaerobic, and shake flask culture conditions were different, the relative flux ratios for some central metabolic reactions did not differ significantly (in particular, between the shake flask and aerobic-chemostat groups). Hence, the central metabolism of S. oneidensis appears to be robust to environmental changes. Our study also demonstrates the merit of coupling GC-MS with 13C NMR for metabolic flux analysis to reduce the use of 13C-labeled substrates and to obtain more-accurate flux values.

Glutamate:Glyoxylate Aminotransferase Modulates Amino Acid Content during Photorespiration

In photorespiration, peroxisomal glutamate:glyoxylate aminotransferase (GGAT) catalyzes the reaction of glutamate and glyoxylate to produce 2-oxoglutarate and glycine. Previous studies demonstrated that alanine aminotransferase-like protein functions as a photorespiratory GGAT. Photorespiratory transamination to glyoxylate, which is mediated by GGAT and serine glyoxylate aminotransferase (SGAT), is believed to play an important role in the biosynthesis and metabolism of major amino acids. To better understand its role in the regulation of amino acid levels, we produced 42 GGAT1 overexpression lines that express different levels of GGAT1 mRNA. The levels of free serine, glycine, and citrulline increased markedly in GGAT1 overexpression lines compared with levels in the wild type, and levels of these amino acids were strongly correlated with levels of GGAT1 mRNA and GGAT activity in the leaves. This accumulation began soon after exposure to light and was repressed under high levels of CO(2). Light and nutrient conditions both affected the amino acid profiles; supplementation with NH(4)NO(3) increased the levels of some amino acids compared with the controls. The results suggest that the photorespiratory aminotransferase reactions catalyzed by GGAT and SGAT are both important regulators of amino acid content.