OCCURRENCE OF GLYCOLATE DEHYDROGENASE AND GLYCOLATE OXIDASE IN SOME COCCOID,ZOOSPORE-PRODUCING GREEN ALGAE1

Twenty-seven species of coccoid, zoospore-producing green algae representing 16 genera in the Chlorococcales and Chlorosarcinales were assayed for glycolate oxidase or glycolate dehydrogenase. Only Planophila terrestris Groover & Bold and Fasciculochloris boldii Trainor, contained glycolate oxidase whereas the others contained glycolate dehydrogenase. Representative algae were grown under varying conditions and assayed to determine any effects on these glycolate enzymes. Although specific rates of enzyme activity often varied widely, the form of glycolate enzyme present was not affected.     

PURIFICATION AND CHARACTERIZATION OF GLYCOLATE OXIDASE FROM THE BROWN ALGA SPATOGLOSSUM PACIFICISM (PHAEOPHYTA)1

Glycolate oxidase was purified to apparent homogeneity from the brown alga Spatoglossum pacificum Yendo. The 1326-fold purified glycolate oxidase enzyme exhibited a specific activity of 22. 4 micromoles glyoxylate formed ·min^?1·mg protein^?1. The molecular weight of the native enzyme was estimated to be 230,000 by gel filtration. The subunit molecular weight of the enzyme was determined to be 49,000 by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, suggesting that the native enzyme is a tetramer. There were two absorption peaks at 345 and 445 nm, indicating that glycolate oxidase is a flavoprotein. This enzyme had a high isoelectric point (pI 9.6) and a high pH optimum (pH 8.3). The K_m values for glycolate and l -lactate were 0.49 and 5.5 mM, respectively. This enzyme also had a broad specificity for other straight-chain α-hydroxy acids but not for β-hydroxyacids. Cyanide, azide, N-ethylmaleimide, and p-chloromercuribenzoic acid did not affect the enzyme, whereas 2-pyridylhydroxymethanesulfonic acid strongly inhibited it. These properties of glycolate oxidase from the brown alga S. pacificum are similar to the properties of the glycolate oxidasesfrom higher plants. Polyclonal antibodies raised against the polypeptide fragment of Spatoglossum glycolate oxidase could recognize glycolate oxidase from Spinacia oleracea L., although the cross-reactivity was weak. The N-terminal sequence of two internal polypeptide fragments of the enzyme from S. pacificum showed a high degree of similarity to that of glycolate oxidase from higher plants. These results suggest that glycolate oxidase from higher plants and brown algae share the same ancestral protein.     

GLYCOLATE DEHYDROGENASE IN PRIMITIVE GREEN ALGAE

Glycolate dehydrogenase occurs in nine species of the Prasinophyceae. The occurrence of glycolate dehydrogenase in these unicellular flagellated organisms does not correspond to the lines of evolution suggested for the green algae based on glycolate enzymology and the nature of the spindle at telophase of mitosis. It is proposed that the evolutionary divergence of the glycolate enzyme came after the evolutionary divergence of the spindle features in the green algae.     

OCCURRENCE OF GLYCOLATE OXIDASE AND HYDROXYPYRUVATE REDUCTASE IN EGREGIA MENZIESII (PHAEOPHYTA)1

Glycolate oxidase and hydroxypyruvate reductase, two key enzymes of glycolate metabolism, are present in the brown alga Egregia menziesii (Turn.) Aresch. The pH optimum and Km for the partially purified glycolate oxidase are similar to the enzyme from higher plants, Charophyta and Xanthophyta, whereas the substrate specificity is different from the higher plant enzyme.     

The occurrence of glycolate dehydrogenase and glycolate oxidase in green plants: an evolutionary survey

Homogenates of various lower land plants, aquatic angiosperms, and green algae were assayed for glycolate oxidase, a peroxisomal enzyme present in green leaves of higher plants, and for glycolate dehydrogenase, a functionally analogous enzyme characteristic of certain green algae. Green tissues of all lower land plants examined (including mosses, liverworts, ferns, and fern allies), as well as three freshwater aquatic angiosperms, contained an enzyme resembling glycolate oxidase, in that it oxidized l- but not d-lactate in addition to glycolate, and was insensitive to 2 mm cyanide. Many of the green algae (including Chlorella vulgaris, previously claimed to have glycolate oxidase) contained an enzyme resembling glycolate dehydrogenase, in that it oxidized d- but not l-lactate, and was inhibited by 2 mm cyanide. Other green algae had activity characteristic of glycolate oxidase and, accordingly, showed a substantial glycolate-dependent O₂ uptake. It is pointed out that this distribution pattern of glycolate oxidase and glycolate dehydrogenase among the green plants may have phylogenetic significance.     

Peroxisomal enzyme activity in Todea barbara gametophytes and sporophytes

Catalase and glycolate oxidase activity were observed in cultured gametophytes and sporophytes of the fern Todea barbara (L.) Moore. The biochemical characteristics of the glycolate-oxidizing enzyme in both plants indicates it is a glycolate oxidase. The results suggest that these plants are capable of photorespiration by a process similar to that occurring in leaves of higher plants.     

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.     

植物中草酸积累与光呼吸乙醇酸代谢的关系

对几种C3 和C4 植物中草酸含量及相应的乙醇酸氧化酶活性测定结果表明 :叶片光呼吸强度及其关键酶活性大小与草酸积累量没有相关性 ;植物根中均能积累草酸 ,但未测出乙醇酸氧化酶活性。烟草根、叶中的草酸含量在不同生长时期差异明显 ,且二者呈极显著正相关 (y =2 .5 6 5lnx 2 .137,r =0 .749,P <0 .0 0 1) ,说明根中草酸可能来自叶片。氧化乙醇酸的酶的活性与氧化乙醛酸的酶的活性呈极显著线性正相关 (y =0 .2 41x 0 .0 0 6 ,r=0 .96 7,P <0 .0 0 0 1) ,进一步证实是乙醇酸氧化酶催化了两种底物的反应。烟草在不同生长期叶片中草酸总含量变化与相应的乙醇酸氧化酶活性变化亦没有相关性 ;低磷胁迫可显著诱导烟草根叶中的草酸形成和分泌 ,但并未影响乙醇酸氧化酶活性 ,进一步证明草酸积累与该酶活性大小无关     

光和底物对乙醇酸氧化酶的基因表达的诱导

从菠菜中提纯了乙醇酸氧化酶并制备其抗体,经免疫双扩散、Ｗｅｓｔｅｒｎｂｌｏｔ和Ｎｏｒｔｈｅｒｎｂｌｏｔ证实水稻和豌豆黄化苗中不存在乙醇酸氧化酶。在黑暗中,底物可促进该酶基因的表达,而在黄化苗光照初期,推测光可能是不经过底物促进该酶基因的表达。     

A novel glycolate oxidase requiring flavin mononucleotide as the cofactor in the prasinophycean alga Mesostigma viride

A new type of glycolate-oxidizing enzyme was found in the prasinophycean alga Mesostigma viride. This enzyme was characterized as the glycolate oxidase that strongly requires FMN, thus distinguishing it from most other Prasinophytes that possess glycolate dehydrogenase instead.     

Isolation and characterization of peroxisomes from diatoms

Peroxisomes were isolated by gradient centrifugation from two different diatoms: Nitzschia laevis (subgroup of Pennales) and Thalassiosira fluviatilis (subgroup of Centrales). In neither of these organelles could catalase or any H₂O₂-forming oxidase be demonstrated. The glycolate-oxidizing enzyme present in the peroxisomes is a dehydrogenase capable of oxidizing l-lactate as well. The peroxisomes also contain the glyoxysomal markers isocitrate lyase and malate synthase. However, enzymes of the fatty-acid -oxidation pathway are located exclusively in the mitochondria. The mitochondria additionally possess glutamate-glyoxylate aminotransferase and a glycolate dehydrogenase which differs from the peroxisomal glycolate dehydrogenase since it preferably utilizes d-lactate as an alternative substrate. Hydroxypyruvate reductase and glyoxylate carboligase were not found in the cells of either diatom. By culturing Nitzschia laevis it could be demonstrated that decreasing the CO₂ concentration in the aeration mixture from 2% to 0.03% and increasing the irradiance from 40 to 250 mol quanta · m^–2 · s^–1 resulted in an increase of all peroxisomal enzyme activities. In addition, enzyme activities of the -oxidation pathway were increased. However, mitochondrial glycolate dehydrogenase and aminotransferase did not alter their activities under these conditions. Summarizing all results, it is postulated that there are two different pathways for the metabolism of glycolate in the diatoms.This work was supported by the Deutsche Forschungsgemeinschaft.     

Glycolate oxidase isoforms are distributed between the bundle sheath and mesophyll tissues of maize leaves

Glycolate oxidase (EC 1.1.3.15) activity was detected both in the bundle sheath (79%) and mesophyll (21%) tissues of maize leaves. Three peaks of glycolate oxidase activity were separated from maize leaves by the linear KCl gradient elution from the DEAE-Toyopearl column. The first peak corresponded to the glycolate oxidase isoenzyme located in the bundle sheath cells, the second peak had a dual location and the third peak was related to the mesophyll fraction. The mesophyll isoenzyme showed higher affinity for glycolate (Km 23 micromol x L(-1)) and a higher pH optimum (7.5-7.6) as compared to the bundle sheath isoenzyme (Km 65 micromol x L(-1), pH optimum 7.3). The bundle sheath isoenzyme was strongly activated by isocitrate and by succinate while the mesophyll isoenzyme was activated by isocitrate only slightly and was inhibited by succinate. It is concluded that although the glycolate oxidase activity is mainly attributed to the bundle sheath, conversion of glycolate to glyoxylate occurs also in the mesophyll tissue of C4 plant leaves.     

Identification and Characterization of Glycolate Oxidase and Related Enzymes from the Endocyanotic Alga Cyanophora paradoxa and from Pea Leaves

Glycolate oxidase (GO) has been identified in the endocyanom Cyanophora paradoxa which has peroxisome-like organelles and cyanelles instead of chloroplasts. The enzyme used or formed equimolar amounts of O₂ or H₂O₂ and glyoxylate, respectively. Aerobically, the enzyme did not reduce the artificial electron acceptor dichlorophenol indophenol. However, after an inhibitor of glycolate dehydrogenase, KCN (2 millimolar), was added to the assay medium, considerable aerobic glycolate:dichlorophenol indophenol reductase activity was detectable. The leaf GO inhibitor 2-hydroxybutynoate (30 micromolar), which binds irreversibly to the flavin moiety of the active site of leaf GO, inhibited Cyanophora GO and pea (Pisum sativum L.) GO to the same extent. This suggests that the active sites of both enzymes are similar. Cyanophora GO and pea GO cannot oxidize d-lactate. In contrast to GO from pea or other organisms, the affinity of Cyanophora GO for l-lactate is very low (K_m 25 millimolar). Another important difference is that Cyanophora GO produced sigmoidal kinetics with O₂ as varied substrate, whereas pea GO produced normal Michaelis-Menten kinetics. It is concluded that there is considerable inhomogeneity among the glycolate-oxidizing enzymes from Cyanophora, pea, and other organisms. The specific catalase activity in Cyanophora was only one-tenth of that in leaves. NADH-and NADPH-dependent hydroxypyruvate reductase (HPR) and glyoxylate reductase activities were detected in Cyanophora. NADH-HPR was markedly inhibited by hydroxypyruvate above 0.5 millimolar. Variable substrate inhibition was observed with glyoxylate in homogenates from different algal cultures. It is proposed that Cyanophora has multiple forms of HPR and glyoxylate reductase, but no enzyme clearly resembling leaf peroxisomal HPR was identified in these homogenates. Moreover, no serine:glyoxylate aminotransferase activity was detected. These results collectively indicate the possibility that the glycolate metabolism in Cyanophora deviates from that in leaves.     

Localization of glycolate dehydrogenase in two species of Dunaliella

The occurrence of glycolate oxidase in addition to glycolate dehydrogenase in Dunaliella salina and D. primolecta, as reported in the literature, could not be confirmed. Both species were demonstrated to possess only glycolate dehydrogenase. After separation of organelles by gradient centrifugation, glycolate dehydrogenase along with hydroxypyruvate reductase was found exclusively in the mitochondria. Thus the peroxisomes from Dunaliella are not of the leaf-type: because of their content of catalase, uricase and hydroxyacyl-CoA dehydrogenase they appear to be of the same type as in Eremosphaera and other chlorophycean algae. No activity of glycolate dehydrogenase was found in the chloroplast fraction when the 2,6-dichlorophenol-indophenol test was used.This work was supported by the Deutsche Forschungsgemeinschaft.     

菠菜叶片乙醇酸氧化酶的氧化甘油酸活性

首先从菠菜叶片中纯化了乙醇酸氧化酶（GO）。通过鉴定反应中氧的消耗以及反应产物H2O2的生成,证实菠菜GO具有氧化光呼吸途径中间代谢物甘油酸的活性。该氧化活性依赖于辅因子FMN和FAD,而不依赖核黄素和光黄素;其最适反应pH值为8．0,Km（甘油酸）值为7．14mmol／L,kcat值为1．04s^-1,活化能为17．29kJ／mol;草酸和丙酮酸对该氧化活性有明显的抑制作用,其中前者为典型的竞争性抑制。进一步通过两底物竞争作图表明：菠菜叶片GO氧化甘油酸反应和氧化乙醇酸反应为同一活性中心所催化。     

Compartmentation of Peroxisomal Enzymes in Algae of the Group of Prasinophyceae : Occurrence of Possible Microbodies without Catalase

Using the Prasinophycean algae Platymonas, Heteromastix, Pedinomonas, and Pyramimonas, subcellular distribution of the enzymes of glycolate metabolism and β-oxidation pathway have been studied. Glycolate dehydrogenase, hydroxypyruvate reductase, and glutamate-glyoxylate aminotransferase are located in the mitochondria. In addition, the mitochondria of all four species contain acyl-coenzyme A (CoA) dehydrogenase, enoyl-CoA hydratase, and thiolase. In Platymonas, Heteromastix, and Pedinomonas, organelles with the characteristic structure of peroxisomes have been detected which also contain the enzymes acyl-CoA oxidase, enoyl-CoA hydratase and thiolase. However, catalase could not be demonstrated in either the peroxisome-like organelles or in the whole cells.     

Enzymes related to lactate metabolism in green algae and lower land plants

Cell-free extracts of Chlorella pyrenoidosa contained two enzymes capable of oxidizing d-lactate; these were glycolate dehydrogenase and NAD(+)-dependent d-lactate dehydrogenase. The two enzymes could be distinguished by differential centrifugation, glycolate dehydrogenase being largely particulate and NAD(+)-d-lactate dehydrogenase being soluble. The reduction of pyruvate by NADH proceeded more rapidly than the reverse reaction, and the apparent Michaelis constants for pyruvate and NADH were lower than for d-lactate and NAD(+). These data indicated that under physiological conditions, the NAD(+)-linked d-lactate dehydrogenase probably functions to produce d-lactate from pyruvate.Lactate dehydrogenase activity dependent on NAD(+) was found in a number of other green algae and in the green tissues of a few lower land plants. When present in species which contain glycolate oxidase rather than glycolate dehydrogenase, the enzyme was specific for l-lactate rather than d-lactate. A cyclic system revolving around the production and utilization of d-lactate in some species and l-lactate in certain others is proposed.     

Cyanobacterial lactate oxidases serve as essential partners in N2 fixation and evolved into photorespiratory glycolate oxidases in plants

Glycolate oxidase (GOX) is an essential enzyme involved in photorespiratory metabolism in plants. In cyanobacteria and green algae, the corresponding reaction is catalyzed by glycolate dehydrogenases (GlcD). The genomes of N(2)-fixing cyanobacteria, such as Nostoc PCC 7120 and green algae, appear to harbor genes for both GlcD and GOX proteins. The GOX-like proteins from Nostoc (No-LOX) and from Chlamydomonas reinhardtii showed high L-lactate oxidase (LOX) and low GOX activities, whereas glycolate was the preferred substrate of the phylogenetically related At-GOX2 from Arabidopsis thaliana. Changing the active site of No-LOX to that of At-GOX2 by site-specific mutagenesis reversed the LOX/GOX activity ratio of No-LOX. Despite its low GOX activity, No-LOX overexpression decreased the accumulation of toxic glycolate in a cyanobacterial photorespiratory mutant and restored its ability to grow in air. A LOX-deficient Nostoc mutant grew normally in nitrate-containing medium but died under N(2)-fixing conditions. Cultivation under low oxygen rescued this lethal phenotype, indicating that N(2) fixation was more sensitive to O(2) in the Δlox Nostoc mutant than in the wild type. We propose that LOX primarily serves as an O(2)-scavenging enzyme to protect nitrogenase in extant N(2)-fixing cyanobacteria, whereas in plants it has evolved into GOX, responsible for glycolate oxidation during photorespiration.     

Developmental studies on microbodies in wheat leaves : I. Conditions influencing enzyme development

Catalase, glycolate oxidase, and hydroxypyruvate reductase, enzymes which are located in the microbodies of leaves, show different developmental patterns in the shoots of wheat seedlings. Catalase and hydroxypyruvate reductase are already present in the shoots of ungerminated seeds. Glycolate oxidase appears later. All three enzymes develop in the dark, but glycolate oxidase and hydroxypyruvate reductase have only low activities. On exposure of the seedlings to continuous white light (14.8 × 10³ ergs cm⁻² sec⁻¹), the activity of catalase is doubled, and glycolate oxidase and hydroxypyruvate reductase activities increase by 4- to 7-fold. Under a higher light intensity, the activities of all three enzymes are considerably further increased. The activities of other enzymes (cytochrome oxidase, fumarase, glucose-6-phosphate dehydrogenase) are unchanged or only slightly influenced by light. After transfer of etiolated seedlings to white light, the induced increase of total catalase activity shows a much longer lag-phase than that of glycolate oxidase and hydroxypyruvate reductase. It is concluded that the light-induced increases of the microbody enzymes are due to enzyme synthesis. The light effect on the microbody enzymes is independent of chlorophyll formation or the concomitant development of functional chloroplasts. Short repeated light exposures which do not lead to greening are very effective. High activities of glycolate oxidase and hydroxypyruvate reductase develop in the presence of 3-amino-1,2,4-triazole which blocks chloroplast development. The effect of light is not exerted through induced glycolate formation and appears instead to be photomorphogenetic in character.     

Experimental evolution of a metabolic pathway for ethylene glycol utilization by Escherichia coli.

Spontaneous mutants of Escherichia coli able to grow on ethylene glycol as a sole source of carbon and energy were obtained from mutants that could grow on propylene glycol. Attempts to obtain ethylene glycol-utilizing mutants from wild-type E. coli were unsuccessful. The two major characteristics of the ethylene glycol-utilizing mutants were (i) increased activities of propanediol oxidoreductase, an enzyme present in the parental strain (a propylene glycol-positive strain), which also converted ethylene glycol into glycolaldehyde; and (ii) constitutive synthesis of high activities of glycolaldehyde dehydrogenase, which converted glycolaldehyde to glycolate. Glycolate was metabolized via the glycolate pathway, which was present in the wild-type cells; this was indicated by the induction in ethylene glycol-grown cells of glycolate oxidase, the first enzyme in the pathway. Glycolaldehyde dehydrogenase was partially characterized as an enzyme of this new metabolic pathway in E. coli, and glycolate was identified as the product of the reaction. This enzyme used NAD and NADP as coenzymes, although the NADP-dependent activity was about 10 times lower than the NAD-dependent activity. Uptake of [14C]ethylene glycol was dependent on the presence of the enzymes capable of metabolism of ethylene glycol. Glycolaldehyde and glycolate were identified as intermediate metabolites in the pathway.