Solubilization, partial purification, and reconstitution of the glycolate/glycerate transporter from chloroplast inner envelope membranes

The glycolate/glycerate transporter of spinach (Spinacia oleracea L.) chloroplast inner envelope membranes was solubilized by treatment of the membranes with sodium cholate. Mixtures of the cholate extracts and soy asolectin were subjected to gel filtration to remove the detergent. The reconstituted vesicles were frozen, thawed, and sonicated in a buffer that contained 10 millimolar d-glycerate and, usually, [³H]sucrose as an internal space indicator. The dilution of the vesicles into a medium that contained 0.4 millimolar [¹⁴C]d-glycerate resulted in a rapid accumulation of labeled glycerate, followed by a much slower loss of [¹⁴C]d-glycerate from the vesicles. This behavior is characteristic of counterflow. The accumulation of [¹⁴C]d-glycerate was strongly inhibited by HgCl₂, which blocks glycolate/glycerate transport in intact chloroplasts. In the absence of proton ionophores, the extent of [¹⁴C]glycolate accumulation under similar conditions was much greater than that of [¹⁴C]d-glycerate. External glycolate inhibited d-glycerate counterflow and external d-glycerate inhibited glycolate counterflow. The external pH dependence of the efflux of [¹⁴C]d-glycerate accumulated in vesicles by counterflow and its inhibition by external l-mandelate are characteristics displayed by glycolate transport in intact chloroplasts. Partial purification of the transporter was achieved by glycerol gradient centrifugation. The solubilized glycolate and glycerate counterflow activities, assayed by reconstitution into vesicles, were found to sediment similarly.     

Chemical inhibition of the glycolate pathway in soybean leaf cells

Isolated soybean (Glycine max [L.] Merr.) leaf cells were treated with three inhibitors of the glycolate pathway in order to evaluate the potential of such inhibitors for increasing photosynthetic efficiency. Preincubation of cells under acid conditions in α-hydroxypyridinemethanesulfonic acid increased ¹⁴CO₂ incorporation into glycolate, but severely inhibited photosynthesis. Isonicotinic acid hydrazide (INH) increased the incorporation of ¹⁴CO₂ into glycine and reduced label in serine, glycerate, and starch. Butyl 2-hydroxy-3-butynoate (BHB) completely and irreversibly inhibited glycolate oxidase and increased the accumulation of ¹⁴C into glycolate. Concomitant with glycolate accumulation was the reduction of label in serine, glycerate, and starch, and the elimination of label in glycine. The inhibitors INH and BHB did not eliminate serine synthesis, suggesting that some serine is synthesized by an alternate pathway. The per cent incorporation of ¹⁴CO₂ into glycolate by BHB-treated cells or glycine by INH-treated cells was determined by the O₂/CO₂ ratio present during assay. Photosynthesis rate was not affected by INH or BHB in the absence of O₂, but these compounds increased the O₂ inhibition of photosynthesis. This finding suggests that the function of the photorespiratory pathway is to recycle glycolate carbon back into the Calvin cycle, so if glycolate metabolism is inhibited, Calvin cycle intermediates become depleted and photosynthesis is decreased. Thus, chemicals which inhibit glycolate metabolism do not reduce photorespiration and increase photosynthetic efficiency, but rather exacerbate the problem of photorespiration.     

Glycolate pathway in green algae

By three criteria, the glycolate pathway of metabolism is present in unicellular green algae. Exogenous glycolate-1-¹⁴C was assimilated and metabolized to glycine-1-¹⁴C and serine-1-¹⁴C. During photosynthetic ¹⁴CO₂ fixation the distributions of ¹⁴C in glycolate and glycine were similar enough to suggest a product-precursor relationship. Five enzymes associated with the glycolate pathway were present in algae grown on air. These were P-glycolate phosphatase, glycolate dehydrogenase (glycolate:dichloroindophenol oxidoreductase), l-glutamate:glyoxylate aminotransferase, serine hydroxymethylase, and glycerate dehydrogenase. Properties of glycerate dehydrogenase and the aminotransferase were similar to those from leaf peroxisomes. The specific activity of glycolate dehydrogenase and serine hydroxymethylase in algae was 1/5 to 1/10 that of the other enzymes, and both these enzymes appear ratelimiting for the glycolate pathway.     

Steady-state photosynthesis in alfalfa leaflets: effects of carbon dioxide concentration

When the CO₂ concentration to which Medicago sativa L. var. El Unico leaflets were exposed was increased from half-saturation to saturation (doubled rate of photosynthesis), glycolate and glycine production apparently decreased due to inhibition of a portion of the glycolate pathway. Serine and glycerate production was not inhibited. We conclude that serine and glycerate were made from 3-phosphoglycerate and not from glycolate and that the conversion of glycine to serine may not be the major source of photorespiratory CO₂ in alfalfa. In investigations of glycolate and photorespiratory metabolism, separate labeling data should be obtained for glycine and serine as those two amino acids may be produced from different precursors and respond differently to environmental perturbations. The increased photosynthetic rate (at saturating CO₂) resulted in greater labeling of both soluble and insoluble products. Sucrose labeling increased sharply, but there was no major shift of tracer carbon flow into sucrose relative to other metabolites. The flow of carbon from the reductive pentose phosphate cycle into the production of tricarboxylic acid cycle intermediates and amino acids increased. Only small absolute increases occurred in steady-state pool sizes of metabolites of the reductive pentose phosphate cycle at elevated CO₂, providing further evidence that the cycle is well regulated.     

Bending of Protonema Cells in a Plastid Glycolate/Glycerate Transporter Knockout Line of Physcomitrella patens

Arabidopsis LrgB (synonym PLGG1) is a plastid glycolate/glycerate transporter associated with recycling of 2-phosphoglycolate generated via the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). We isolated two homologous genes (PpLrgB1 and B2) from the moss Physcomitrella patens. Phylogenetic tree analysis showed that PpLrgB1 was monophyletic with LrgB proteins of land plants, whereas PpLrgB2 was divergent from the green plant lineage. Experiments with PpLrgB–GFP fusion proteins suggested that both PpLrgB1 and B2 proteins were located in chloroplasts. We generated PpLrgB single (∆B1 and ∆B2) and double (∆B1/∆B2)-knockout lines using gene targeting of P. patens. The ∆B1 plants showed decreases in growth and photosynthetic activity, and their protonema cells were bent and accumulated glycolate. However, because ∆B2 and ∆B1/∆B2 plants showed no obvious phenotypic change relative to the wild-type or ∆B1 plants, respectively, the function of PpLrgB2 remains unclear. Arabidopsis LrgB could complement the ∆B1 phenotype, suggesting that the function of PpLrgB1 is the same as that of AtLrgB. When ∆B1 was grown under high-CO₂ conditions, all novel phenotypes were suppressed. Moreover, protonema cells of wild-type plants exhibited a bending phenotype when cultured on media containing glycolate or glycerate, suggesting that accumulation of photorespiratory metabolites caused P. patens cells to bend.     

Localization of glycerate kinase and some enzymes for sucrose synthesis in c(3) and c(4) plants

The localization of some key enzymes leading to sucrose synthesis in photosynthetic tissue of C₃ and C₄ species was investigated. These included UDP-glucose (UDPG) pyrophosphorylase, sucrose phosphate synthetase, and glycerate kinase. Whether glycerate kinase is localized exclusively in the chloroplast or partly outside the chloroplast could influence the fate of carbon flow to sucrose through the glycolate pathway.     

Chloroplast phosphofructokinase in the green alga, Dunaliella marina: partial purification and kinetic and regulatory properties

The aim of this work was to study the pathway(s) of sugar phosphate metabolism in chloroplasts of the unicellular green alga, Dunaliella marina (Volvocales). Phosphofructokinase, detectable in crude cell extracts, copurifled with intact chloroplasts on sucrose density gradients. In isolated chloroplasts, phosphofructokinase activity displayed latency to the same degree as chloroplast marker enzymes. From the quantitative distribution of enzyme activities in fractionated cells, it is concluded that there is an exclusive localization of phosphofructokinase in chloroplasts. In addition, no separation into multiple forms could be achieved. For the study of regulatory properties, chloroplast phosphofructokinase was partially purified by ammonium sulfate fractionation followed by DEAE-cellulose chromatography. The pH optimum of the enzyme activity was 7.0 and was not altered with varying concentrations of substrates or low-molecular-weight effectors. Fructose 6-phosphate showed a sigmoidal saturation curve whose shape was further changed with varying protein concentrations of the preparation. The second substrate, ATP, gave a hyperbolic saturation curve with a Michaelis constant of 60 μm. At a Mg²⁺ concentration of 2.5 mm, ATP concentrations exceeding 1 mm inhibited the enzyme in a positive cooperative manner. The same type of inhibition was observed with other phosphorylated intermediates of carbon metabolism, the most efficient being phosphoenolpyruvate, glycolate 2-phosphate, glycerate 3-phosphate, and glycerate 2-phosphate. Inorganic phosphate was the only activator found for phosphofructokinase. With nonsaturating fructose 6-phosphate concentrations, P_i activated in a positive cooperative fashion, while no activation occurred with saturating fructose 6-phosphate concentrations. In the presence of either an activator or an inhibitor, the sigmoidal shape of the fructose 6-phosphate saturation curve was altered. Most notably, the activator P_i could relieve the inhibitory action of ATP, phosphoenolpyruvate, glycerate 3-phosphate, glycerate 2-phosphate, and glycolate 2-phosphate. Based on these experimental findings, the regulatory properties of D. marina chloroplast phosphofructokinase are discussed with respect to its playing a key role in the regulation of chloroplast starch metabolism during a light/dark transition. All available evidence is compatible with the interpretation that phosphofructokinase is active only in the dark thus channeling starch degradation products into glycolysis.     

The plant-like C2 glycolate cycle and the bacterial-like glycerate pathway cooperate in phosphoglycolate metabolism in cyanobacteria

The occurrence of a photorespiratory 2-phosphoglycolate metabolism in cyanobacteria is not clear. In the genome of the cyanobacterium Synechocystis sp. strain PCC 6803, we have identified open reading frames encoding enzymes homologous to those forming the plant-like C2 cycle and the bacterial-type glycerate pathway. To study the route and importance of 2-phosphoglycolate metabolism, the identified genes were systematically inactivated by mutagenesis. With a few exceptions, most of these genes could be inactivated without leading to a high-CO(2)-requiring phenotype. Biochemical characterization of recombinant proteins verified that Synechocystis harbors an active serine hydroxymethyltransferase, and, contrary to higher plants, expresses a glycolate dehydrogenase instead of an oxidase to convert glycolate to glyoxylate. The mutation of this enzymatic step, located prior to the branching of phosphoglycolate metabolism into the plant-like C2 cycle and the bacterial-like glycerate pathway, resulted in glycolate accumulation and a growth depression already at high CO(2). Similar growth inhibitions were found for a single mutant in the plant-type C2 cycle and more pronounced for a double mutant affected in both the C2 cycle and the glycerate pathway after cultivation at low CO(2). These results suggested that cyanobacteria metabolize phosphoglycolate by the cooperative action of the C2 cycle and the glycerate pathway. When exposed to low CO(2), glycine decarboxylase knockout mutants accumulated far more glycine and lysine than wild-type cells or mutants with inactivated glycerate pathway. This finding and the growth data imply a dominant, although not exclusive, role of the C2 route in cyanobacterial phosphoglycolate metabolism.     

Subcellular Distribution of Enzymes of Glycolate Metabolism in the Alga Cyanidium caldarium

The intracellular distribution of enzymes capable of catalyzing the reactions from phosphoglycolate to glycerate in the bluegreen colored eucaryotic alga Cyanidium caldarium has been studied. After separating the organelles from a crude homogenate on a linear flotation gradient, the enzymes glycolate oxidase and glutamate-glyoxylate aminotransferase along with catalase were present in the peroxisomal fraction (density: 1.23 grams per cubic centimeter). Serine hydroxymethyltransferase was found in the mitochondrial fraction (density: 1.18 grams per cubic centimeter). In contrast to the observations in green leaves of higher plants, the enzymes for the conversion of serine to glycerate (serine-glyoxylate aminotransferase and hydroxypyruvate reductase) were found only in the soluble fraction of the gradient. The partial characterization of enzymes from Cyanidium participating in glycolate metabolism revealed only slight differences from the corresponding enzymes from higher plants. The phylogenetic implications of the observed similarities between the enigmatic alga Cyanidium and higher plants are discussed.     

Glycolate metabolism and subcellular distribution of glycolate oxidase in Spatoglossum pacificum (Phaeophyceae, Chromophyta)

Seven enzymes participating in glycolate metabolism were demonstrated to be present in crude extract of the brown alga Spatoglossum pacificum Yendo. These were phosphoglycolate phosphatase, glycolate oxidase, glutamate-glyoxylate aminotransferase, serine hydroxymethyltransferase, amino acid-hydroxy-pyruvate aminotransferase, hydroxypyruvate reductase and catalase. Malate synthase, which is involved in glycolate metabolism in the xanthophycean alga, could not be detected. On demonstration of subcellular distribution of glycolate oxidizing enzymes by linear sucrose density gradient centrifugation, glycolate oxidase was detected in the same fraction at a density of 1.23 g cm^?3 with catalase: that is, the marker enzyme of peroxisome and serine hydroxymethyltransferase was found in the same fraction at a density of 1.21 g cm^?3 with isocitrate dehydrogenase, the marker of mitochondria. From the present data, it is proposed that the brown alga Spatoglossum possesses the ability to metabolize glycolate to glycerate via the pathway which may be similar to that of higher plants.     

Only plant-type (GLYK) glycerate kinases produce d-glycerate 3-phosphate

d-Glycerate kinases (GK) occur in three phylogenetically distinct classes. Class II GKs produce glycerate 2-phosphate, while both class I GK and class III GK (GLYK) are thought to produce glycerate 3-phosphate. We report on the identification of a bacterial-type class I GK in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 and of a plant-type GLYK in the filamentous cyanobacterium Nostoc sp. strain PCC 7120. The comparison with other prokaryotic and eukaryotic GKs of both classes shows that glycerate 3-phosphate is produced only by the GLYKs, but, in contrast to current thinking, not by any of the examined class I enzymes.     

Conversion of serine to glycerate in intact spinach leaf peroxisomes: role of malate dehydrogenase

In photorespiration, leaf peroxisomes convert serine to glycerate via serine-glyoxylate aminotransferase and NADH-hydroxypyruvate reductase. We isolated intact spinach leaf peroxisomes in 0.25 M sucrose, and characterized their enzymatic conversion of serine to glycerate using physiological concentrations of substrates and coenzymes. In the presence of glycolate (glyoxylate), and NADH and NAD alone or together in physiological proportions, the rate of serine-to-glycerate conversion was enhanced and sustained by the addition of malate. The rate was similar at 1 and 5 mM serine, but was two to three times higher in 50 mM than 5 mM malate. In the presence of NAD and malate, there was 1:1 stoichiometric formation of glycerate and oxaloacetate. Addition of 1 or 5 mM glutamate resulted in a negligible enhancement of the conversion of hydroxypyruvate to glycerate. Intact peroxisomes produced glycerate from either serine or hydroxypyruvate at a rate two times higher than osmotically lysed peroxisomes. These results suggest that under physiological conditions, the peroxisomal malate dehydrogenase operates independent of aspartate-alpha-ketoglutarate aminotransferase in supplying NADH for hydroxypyruvate reduction. This supply of NADH is the rate-limiting step in the conversion of serine to glycerate. The compartmentation of hydroxypyruvate reductase and malate dehydrogenase in the peroxisomes confers a higher efficiency in the supply of NADH for hydroxypyruvate reduction under a normal, high NAD/NADH ratio in the cytosol.     

A Mathematical Model of Photorespiration and Photosynthesis

A comprehensive mathematical model of C₃ leaf carbon metabolism,involving the Calvin cycle and the glycolate and glycerate pathwaysof photorespiration, is formulated in terms of a system of non-lineardifferential equations. A steady state, which is found to beeffectively stable, is derived. The model behaves realisticallywhen tested under varying external carbon dioxide and oxygenconcentrations: photosynthesis is inhibited by higher oxygenlevels, while photorespiration is inhibited by higher carbondioxide levels. Calvin cycle, differential equations, glycolate pathway, mathematical model, photorespiration, photosynthesis     

Aerobic and anaerobic respiration in the intact spinach chloroplast

Aerobic and anaerobic chloroplastic respiration was monitored by measuring ¹⁴CO₂ evolution from [¹⁴C]glucose in the darkened spinach (Spinacia oleracea) chloroplast and by estimating the conversion of fructose 1,6-bisphosphate to glycerate 3-phosphate in the darkened spinach chloroplast in air with O₂ or in N₂ with nitrite or oxaloacetate as electron acceptors. The pathway of ¹⁴CO₂ evolution from labeled glucose in the absence and presence of the inhibitors iodoacetamide and glycolate 2-phosphate under air or N₂ were those expected from the oxidative pentose phosphate cycle and glycolysis. Of the electron acceptors, O₂ was the best (2.4 nanomoles CO₂ per milligram chlorophyll per hour), followed by nitrite and oxaloacetate. With respect to glycerate 3-phosphate formation from fructose 1,6-bisphosphate, methylene blue increased the aerobic rate from 3.7 to 5.4 micromoles per milligram chlorophyll per hour. A rate of 4.8 micromoles per milligram chlorophyll per hour was observed under N₂ with nitrite and oxaloacetate.     

Chloroplast protein PLGG1 is involved in abscisic acid-regulated lateral root development and stomatal movement in Arabidopsis

The plant hormone abscisic acid (ABA) plays a crucial role in root architecture; however, the molecular mechanism of ABA-regulated lateral root (LR) growth is not well known. We screened an Arabidopsis thaliana mutant with LR growth that was sensitive to ABA from a T-DNA insertion mutant library, which was an allelic mutant of plgg1-1, termed plgg1-2. PLGG1 encodes a chloroplast protein that transports plastidic glycolate and glycerate. The length and number of LRs at the root-hypocotyl junction of plgg1-1 and plgg1-2 were significantly impaired under exogenous ABA treatment, and the transgenic plant complementary lines of plgg1-2 restored LR growth in response to ABA. In addition, we found that PLGG1 is involved in other major ABA responses, including ABA-inhibited seed germination, ABA-mediated stomatal movement, and drought tolerance. These findings open new perspectives on elucidating the mechanism of ABA response, and provide clues for analysing the functions of chloroplast proteins in regulating root growth.     

Lack of ATP requirement for light stimulation of glycerate transport into intact isolated chloroplasts

Light increased the initial rate and the extent of glycerate uptake by intact isolated chloroplasts. Half-maximum stimulation occurred with 10 to 20 watts per square meter of red light. Preillumination of chloroplasts enhanced uptake in a subsequent dark period. The light effect was abolished by DCMU and also by uncoupling agents such as nigericin and carbonyl cyanide p-trifluoromethoxyphenyl hydrazone.

Arsenate and phlorizin only inhibited glycerate uptake to the extent that metabolism in the chloroplast was decreased by insufficient ATP. The concentration of glycerate accumulated in the chloroplast stroma was not significantly decreased. Chloroplasts isolated from young pea shoots (Pisum sativum, L. cv Massey Gem) were depleted of ATP by incubation with inorganic pyrophosphate or with ATP analogs. These treatments also decreased metabolism of glycerate but the actual concentration of glycerate accumulated in the chloroplast stroma was not decreased.

The results indicate that glycerate uptake is driven by ion gradients established across the chloroplast envelope in the light. ATP is not involved in the transport of glycerate into chloroplasts, being required only for the subsequent metabolism of glycerate in the chloroplast stroma. It is proposed that glycerate transport may be coupled to the proton gradient established in the light across the chloroplast envelope.

     

Stereochemical studies on a plasmid-coded fluoroacetate halidohydrolase

The stereochemical course of action of haloacetate halidohydrolase H-1 from Pseudomonas sp., strain A, which catalyzes the dehalogenation of fluoroacetate to glycolate, has been determined by enzymatic analysis of products from incubations with both enantiomers of 20-fluoropropionate, and by ¹H NMR analysis of the ester of (?)-α-methoxy-α-(trifluoromethyl)phenylacetic acid with phenacyl [2-²H₁]glycolate derived from the product of incubation with the (S)-monodeuterofluoroacetate. The results support a direct displacement mechanism for this enzyme, since they indicate that the reaction is catalyzed with inversion of configuration.     

Desensitization of Feedback Inhibition of the Saccharomyces cerevisiae γ-Glutamyl Kinase Enhances Proline Accumulation and Freezing Tolerance

In response to osmotic stress, proline is accumulated in many bacterial and plant cells as an osmoprotectant. The yeast Saccharomyces cerevisiae induces trehalose or glycerol synthesis but does not increase intracellular proline levels during various stresses. Using a proline-accumulating mutant, we previously found that proline protects yeast cells from damage by freezing, oxidative, or ethanol stress. This mutant was recently shown to carry an allele of PRO1 which encodes the Asp154Asn mutant γ-glutamyl kinase (GK), the first enzyme of the proline biosynthetic pathway. Here, enzymatic analysis of recombinant proteins revealed that the GK activity of S. cerevisiae is subject to feedback inhibition by proline. The Asp154Asn mutant was less sensitive to feedback inhibition than wild-type GK, leading to proline accumulation. To improve the enzymatic properties of GK, PCR random mutagenesis in PRO1 was employed. The mutagenized plasmid library was introduced into an S. cerevisiae non-proline-utilizing strain, and proline-overproducing mutants were selected on minimal medium containing the toxic proline analogue azetidine-2-carboxylic acid. We successfully isolated several mutant GKs that, due to extreme desensitization to inhibition, enhanced the ability to synthesize proline better than the Asp154Asn mutant. The amino acid changes were localized at the region between positions 142 and 154, probably on the molecular surface, suggesting that this region is involved in allosteric regulation. Furthermore, we found that yeast cells expressing Ile150Thr and Asn142Asp/Ile166Val mutant GKs were more tolerant to freezing stress than cells expressing the Asp154Asn mutant.     

Glycerate 2-kinase of Thermotoga maritima and genomic reconstruction of related metabolic pathways

Members of a novel glycerate-2-kinase (GK-II) family were tentatively identified in a broad range of species, including eukaryotes and archaea and many bacteria that lack a canonical enzyme of the GarK (GK-I) family. The recently reported three-dimensional structure of GK-II from Thermotoga maritima (TM1585; PDB code 2b8n) revealed a new fold distinct from other known kinase families. Here, we verified the enzymatic activity of TM1585, assessed its kinetic characteristics, and used directed mutagenesis to confirm the essential role of the two active-site residues Lys-47 and Arg-325. The main objective of this study was to apply comparative genomics for the reconstruction of metabolic pathways associated with GK-II in all bacteria and, in particular, in T. maritima. Comparative analyses of ~400 bacterial genomes revealed a remarkable variety of pathways that lead to GK-II-driven utilization of glycerate via a glycolysis/gluconeogenesis route. In the case of T. maritima, a three-step serine degradation pathway was inferred based on the tentative identification of two additional enzymes, serine-pyruvate aminotransferase and hydroxypyruvate reductase (TM1400 and TM1401, respectively), that convert serine to glycerate via hydroxypyruvate. Both enzymatic activities were experimentally verified, and the entire pathway was validated by its in vitro reconstitution.     

Influence of pH upon the Warburg Effect in Isolated Intact Spinach Chloroplasts: II. Interdependency of Glycolate Synthesis upon pH and Calvin Cycle Intermediate Concentration in the Absence of Carbon Dioxide Photoassimilation

The light-dependent synthesis of glycolate derived from fructose 1,6-diphosphate, ribose 5-phosphate, or glycerate 3-phosphate was studied in the intact spinach (Spinacia oleracea) chloroplasts in the absence of CO(2). Glycolate yield increased with an elevation of O(2), pH, and the concentration of the phosphorylated compound supplied. No pH optimum was observed as the pH was increased from 7.4 to 8.5. The average maximal rate of glycolate synthesis was 50 mumoles per milligram chlorophyll per hour while the highest rate observed was 92 with 2.5 mm fructose 1,6-diphosphate in 100% O(2). The highest yields of glycolate synthesized from fructose 1,6-diphosphate, ribose 5-phosphate, or glycerate 3-phosphate were 0.14, 0.24, and 0.30, respectively, on a molar basis.