Fermentative degradation of glyoxylate by a new strictly anaerobic bacterium

A new strictly anaerobic, gram-negative, nonsporeforming bacterium, Strain PerGlx1, was enriched and isolated from marine sediment samples with glyoxylate as sole carbon and energy source. The guanineplus-cytosine content of the DNA was 44.1±0.2 mol %. Glyoxylate was utilized as the only substrate and was stoichiometrically degraded to carbon dioxide, hydrogen, and glycolate. An acetyl-CoA and ADP-dependent glyoxylate converting enzyme activity, malic enzyme, and pyruvate synthase were found at activities sufficient for growth (0.25 U x mg protein^-1). These findings allow to design a new degradation pathway for glyoxylate: glyoxylate is condensed with acetyl-CoA to form malyl-CoA; the free energy of the thioester linkage in malyl-CoA is conserved by substrate level phosphorylation. Part of the electrons released during glyoxylate oxidation to CO₂ reduce a small fraction of glyoxylate to glycolate.     

Evidence for the presence in tobacco leaves of multiple enzymes for the oxidation of glycolate and glyoxylate

The enzymic oxidation of glycolate to glyoxylate and glyoxylate to oxalate by preparations purified from tobacco (Nicotiana tabacum var Havana Seed) leaves was studied. The K_m values for glycolate and glyoxylate were 0.26 and 1.0 millimolar, respectively. The ratio of glycolate to glyoxylate oxidation was 3 to 4 in crude extracts but decreased to 1.2 to 1.5 on purification by (NH₄)₂SO₄ fractionation and chromatography on agarose A-15 and hydroxylapatite. This level of glyoxylate oxidation activity was higher than that previously found for glycolate oxidase (EC 1.1.3.1). The ratio of the two activities was changed by reaction with the substrate analog 2-hydroxy-3-butynoate (HBA) which at all concentrations inhibited glyoxylate oxidation to a greater extent than glycolate oxidation. The ratio of the two activities could also be altered by changing the O₂ concentration. Glycolate oxidation increased 3.6-fold when the O₂ atmosphere was increased from 21 to 100%, whereas glyoxylate oxidation increased only 1.6-fold under the same conditions. These changes in ratio during purification, on inhibition by HBA, and under varying O₂ concentrations imply that tobacco leaves contain at least two enzymes capable of oxidizing glycolate and glyoxylate.     

荞麦与大豆叶片乙醇酸氧化酶的纯化和性质比较研究

分别从荞麦与大豆叶片中部分纯化了乙醇酸氧化酶 (GO ,EC1 .1 .3 .1 ) ,并研究其部分性质。结果显示荞麦与大豆叶片中GO的催化特性有明显差异 :大豆叶片中GO对乙醇酸Km值为 0 .3 1mmol/L ,对乙醛酸Km值为 1 .98mmol/L。外源草酸对GO氧化乙醇酸活性影响很小 ,但对其氧化乙醛酸活性抑制明显 ,5mmol/L草酸可抑制 44%。而荞麦叶片中GO性质有所不同 :GO对乙醇酸Km为 0 .46mmol/L ,对乙醛酸Km为 0 .85mmol/L。草酸对荞麦GO氧化乙醇酸活性影响也很小 ,对其氧化乙醛酸活性的抑制作用明显小于大豆 ,5mmol/L草酸只抑制 2 4%。上述研究结果表明 ,荞麦GO对乙醛酸的亲和力明显强于大豆 ,并且草酸对其GO氧化乙醛酸活性影响较小。因此相对于大豆而言 ,GO可能在荞麦叶片草酸合成中起重要作用。     

Oxidation of Ethylene Glycol by a Salt-Requiring Bacterium

Bacterium T-52, cultured on ethylene glycol, readily oxidized glycolate and glyoxylate and exhibited elevated activities of ethylene glycol dehydrogenase and glycolate oxidase. Labeled glyoxylate was identified in reaction mixtures containing [¹⁴C]-ethylene glycol, but no glycolate was detected. The most likely pathway of ethylene glycol catabolism by bacterium T-52 is sequential oxidation to glycolate and glyoxylate.     

Metabolism of glycolate and glyoxylate in intact spinach leaf peroxisomes

Intact and broken (osmotically disrupted) spinach (Spinacia oleracea) leaf peroxisomes were compared for their enzymic activities on various metabolites in 0.25 molar sucrose solution. Both intact and broken peroxisomes had similar glycolate-dependent o₂ uptake activity. In the conversion of glycolate to glycine in the presence of serine, intact peroxisomes had twice the activity of broken peroxisomes at low glycolate concentrations, and this difference was largely eliminated at saturating glycolate concentrations. However, when glutamate was used instead of serine as the amino group donor, broken peroxisomes had slightly higher activity than intact peroxisomes. In the conversion of glyoxylate to glycine in the presence of serine, intact peroxisomes had only about 50% of the activity of broken peroxisomes at low glyoxylate concentrations, and this difference was largely overcome at saturating glyoxylate concentrations. In the transamination between alanine and hydroxypyruvate, intact peroxisomes had an activity only slightly lower than that of broken peroxisomes. In the oxidation of NADH in the presence of hydroxypyruvate, intact peroxisomes were largely devoid of activity. These results suggest that the peroxisomal membrane does not impose an entry barrier to glycolate, serine, and O₂ for matrix enzyme activity; such a barrier does exist to glutamate, alanine, hydroxypyruvate, glyoxylate, and NADH. Furthermore, in intact peroxisomes, glyoxylate generated by glycolate oxidase is channeled directly to glyoxylate aminotransferase for a more efficient glycolate-glycine conversion. In related studies, application of in vitro osmotic stress to intact or broken peroxisomes had little effect on their ability to metabolize glycolate to glycine.     

Role of Glycine and Glyoxylate Decarboxylation in Photorespiratory CO(2) Release

Mechanically isolated soybean leaf cells metabolized added glycolate by two mechanisms, the direct oxidation of glyoxylate and the decarboxylation of glycine. The rate of glyoxylate oxidation was dependent on the cellular glyoxylate concentration and was linear between 0.58 and 2.66 micromoles glyoxylate per milligram chlorophyll. The rate extrapolated to zero at a concentration of zero. The concentration and, therefore, the rate of oxidation of glyoxylate could be decreased by adding glutamate or serine to the cells. These substrates were amino donors for the transamination of glyoxylate to glycine. In the presence of these amino acids more CO₂ was released from added glycolate via the glycine decarboxylation reaction and less by the direct oxidation of glyoxylate.     

Glyoxylate intermediate of the direct oxidation of acetate and glycolate byE. coli

Summary From the oxidation of malate and citrate (instead of isocitrate) by livingE. coli cells no formation of glyoxylate could be observed. On the other hand glyoxylate was always formed in the presence of acetate or glycolate. This result is further proof that glyoxylate is a direct intermediate of the oxidation of acetate and glycolate according to the monocarboxylic acid scheme.     

The formation of oxalate from glycolate in rat and human liver

In this study, we attempted to elucidate the metabolic pathway and enzymes actually involved in oxalate formation from glycolate in rat and human liver. In rat liver, the formation of oxalate from glycolate appeared to take place predominantly via glyoxylate. The oxalate formation from glycolate observed with crude enzyme preparations was almost entirely accounted for by the sequential actions of glycolate oxidase and xanthine oxidase (XOD) or lactate dehydrogenase (LDH). Under the conditions used, no significant activity was attributable to glycolate dehydrogenase, an enzyme reported to catalyze the direct oxidation of glycolate to oxalate. Among the three enzymes known to catalyze the oxidation of glyoxylate to oxalate, glycolate oxidase and XOD showed much lower activities (a higher Km and lower Vmax) toward glyoxylate than those with the respective primary substrates. As to LDH, none of the LDH subunit-deficient patients examined showed profoundly lowered urinary oxalate excretion. Based on the results obtained, the presumed efficacies in vivo of individual enzymes, as catalysts of glyoxylate oxidation, and the in vivo conditions assumed to allow their catalysis of oxalate production are discussed.     

The inhibition of oxalate biosynthesis in isolated perfused rat liver by DL-phenyllactate and n-heptanoate

Glycolate oxidase was isolated and partially purified from human and rat liver. The enzyme preparation readily catalyzed the oxidation of glycolate, glyoxylate, lactate, hydroxyisocaproate and α-hydroxybutyrate. The oxidation of glycolate and glyoxylate by glycolate oxidase was completely inhibited by 0.02 m dl-phenyllactate or n-heptanoate. The oxidation of glyoxylate by lactic dehydrogenase or xanthine oxidase was not inhibited by 0.067 m dl-phenyllactate or n-heptanoate. The conversion of [U-¹⁴C] glyoxylate to [¹⁴C] oxalate by isolated perfused rat liver was completely inhibited by dl-phenyllactate and n-heptanoate confirming the major contribution of glycolate oxidase in oxalate synthesis. Since the inhibition of oxalate was 100%, lactic dehydrogenase and xanthine oxidase do not contribute to oxalate biosynthesis in isolated perfused rat liver. dl-Phenyllactate also inhibited [¹⁴C] oxalate synthesis from [1-¹⁴C] glycolate, [U-¹⁴C] ethylene glycol, [U-¹⁴C] glycine, [3-¹⁴C] serine, and [U-¹⁴C] ethanolamine in isolated perfused rat liver. Oxalate synthesis from ethylene glycol was inhibited by dl-phenyllactate in the intact male rat confirming the role of glycolate oxidase in oxalate synthesis in vivo and indicating the feasibility of regulating oxalate metabolism in primary hyperoxaluria, ethylene glycol poisoning, and kidney stone formation by enzyme inhibitors.     

Active site and loop 4 movements within human glycolate oxidase: implications for substrate specificity and drug design

Human glycolate oxidase (GO) catalyzes the FMN-dependent oxidation of glycolate to glyoxylate and glyoxylate to oxalate, a key metabolite in kidney stone formation. We report herein the structures of recombinant GO complexed with sulfate, glyoxylate, and an inhibitor, 4-carboxy-5-dodecylsulfanyl-1,2,3-triazole (CDST), determined by X-ray crystallography. In contrast to most alpha-hydroxy acid oxidases including spinach glycolate oxidase, a loop region, known as loop 4, is completely visible when the GO active site contains a small ligand. The lack of electron density for this loop in the GO-CDST complex, which mimics a large substrate, suggests that a disordered to ordered transition may occur with the binding of substrates. The conformational flexibility of Trp110 appears to be responsible for enabling GO to react with alpha-hydroxy acids of various chain lengths. Moreover, the movement of Trp110 disrupts a hydrogen-bonding network between Trp110, Leu191, Tyr134, and Tyr208. This loss of interactions is the first indication that active site movements are directly linked to changes in the conformation of loop 4. The kinetic parameters for the oxidation of glycolate, glyoxylate, and 2-hydroxy octanoate indicate that the oxidation of glycolate to glyoxylate is the primary reaction catalyzed by GO, while the oxidation of glyoxylate to oxalate is most likely not relevant under normal conditions. However, drugs that exploit the unique structural features of GO may ultimately prove to be useful for decreasing glycolate and glyoxylate levels in primary hyperoxaluria type 1 patients who have the inability to convert peroxisomal glyoxylate to glycine.     

Electron transport phosphorylation driven by glyoxylate respiration with hydrogen as electron donor in membrane vesicles of a glyoxylate-fermenting bacterium

The syntrophically glycolate-fermenting bacterium in the methanogenic binary coculture FlGlyM was isolated in pure culture (strain FlGlyR) with glyoxylate as sole substrate. This strain disproportionated 12 glyoxylate to 7 glycolate, 10 CO₂, and 3 hydrogen. Glyoxylate was oxidized via the malyl-CoA pathway. All enzymes of this pathway, i.e. malyl-CoA lyase/malate: CoA ligase, malic enzyme, and pyruvate synthase, were demonstrated in cell-free extracts. Glycolate dehydrogenase, hydrogenase, and ATPase, as well as menaquinones as potential electron carriers, were present in the membranes. Everted membrane vesicles catalyzed hydrogen-dependent glyoxylate reduction to glycolate [86–207 nmol min^-1 (mg protein)^-1] coupled to ATP synthesis from ADP and P_i [38–82 nmol min^-1 (mg protein)^-1]. ATP synthesis was abolished entirely by protonophores or ATPase inhibitors (up to 98 and 94% inhibition, respectively) indicating the involvement of proton-motive force in an electron transport phosphorylation driven by a new glyoxylate respiration with hydrogen as electron donor. Measured reaction rates in vesicle preparations revealed a stoichiometry of ATP formation of 0.2–0.5 ATP per glyoxylate reduced.Abbreviations BES 2-Bromoethanesulfonate - CCCP Carbonylcyanide m-chlorophenylhydrazone - DCCD N,N-Dicyclohexylcarbodiimide - DCPIP 2,6-Dichlorophenolindophenol - DTE Dithioerythritol - TCS 3,5,4,5-Tetrachlorosalicylanilide - SF 6847 3,5-Di-tert-butyl-4-hydroxybenzylidenemalonitrile     

菜心乙醇酸氧化酶的纯化和催化特性分析

用改进后的方法,从菜心绿叶中分离纯化得到一个亚基分子量为42kD的乙醇酸氧化酶,用氧电极法测定该酶同时能催化乙醇酸和乙醛酸的氧化。     

Glycolate and glyoxylate metabolism in HepG2 cells

Oxalate synthesis in human hepatocytes is not well defined despite the clinical significance of its overproduction in diseases such as the primary hyperoxalurias. To further define these steps, the metabolism to oxalate of the oxalate precursors glycolate and glyoxylate and the possible pathways involved were examined in HepG2 cells. These cells were found to contain oxalate, glyoxylate, and glycolate as intracellular metabolites and to excrete oxalate and glycolate into the medium. Glycolate was taken up more effectively by cells than glyoxylate, but glyoxylate was more efficiently converted to oxalate. Oxalate was formed from exogenous glycolate only when cells were exposed to high concentrations. Peroxisomes in HepG2 cells, in contrast to those in human hepatocytes, were not involved in glycolate metabolism. Incubations with purified lactate dehydrogenase suggested that this enzyme was responsible for the metabolism of glycolate to oxalate in HepG2 cells. The formation of ¹⁴C-labeled glycine from ¹⁴C-labeled glycolate was observed only when cell membranes were permeabilized with Triton X-100. These results imply that peroxisome permeability to glycolate is restricted in these cells. Mitochondria, which produce glyoxylate from hydroxyproline metabolism, contained both alanine:glyoxylate aminotransferase (AGT)2 and glyoxylate reductase activities, which can convert glyoxylate to glycine and glycolate, respectively. Expression of AGT2 mRNA in HepG2 cells was confirmed by RT-PCR. These results indicate that HepG2 cells will be useful in clarifying the nonperoxisomal metabolism associated with oxalate synthesis in human hepatocytes. liver; peroxisomes; hepatocytes; hyperoxaluria; alanine:glyoxylate aminotransferase; glyoxylate reductase     

Rates of glycolate synthesis and metabolism during photosynthesis of Euglena and microalgae grown on low CO2

The rate of glycolate excretion in Euglena gracilis Z and some microalgae grown at the atmospheric level of CO₂ was determined using amino-oxyacetate (AOA). The extracellular O₂ concentration was kept at 240 M by bubbling the incubation medium with air. Glycolate, the main excretion product, was excreted by Euglena at 6 mol·h^-1·(mg chlorophyll (Chl))^-1. Excretion depended on the presence of AOA, and was saturated at 1 mM AOA. A substituted oxime formed from glyoxylate and AOA was also excreted. Bicarbonate added at 0.1 mM did not prevent the excretion of glycolate. The excretion of glycolate increased with higher O₂ concentrations in the medium, and was competitively inhibited by much higher concentrations of bicarbonate. Aminooxyacetate also caused excretion of glycolate from the green algae, Chlorella pyrenoidosa, Scenedesmus obliquus and Chlamydomonas reinhardtii grown on air, at the rates of 2–7 mol·h^-1·(mg Chl)^-1 in the presence of 0.2–0.6 mM dissolved inorganic carbon, but the cyanobacterium, Anacystis nidulans, grown in the same way did not excrete glycolate. The efficiency of the CO₂-concentrating mechanism to suppress glycolate formation is discussed on the basis of the magnitude of glycolate formation in these low-CO₂-grown cells.Abbreviations AOA aminooxyacetate - Chl chlorophyll - DIC dissolved inorganic carbon - HPLC high-pressure liquid chromatography - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase This is the 16th paper in a series on the metabolism of glycolate in Euglena gracilis. The 15th paper is Yokota et al. (1985c)     

The synthesis of oxalate from hydroxypyruvate by isolated perfused rat liver the mechanism of hyperoxaluria in L-lyceric aciduria

Hydroxypyruvate and glycolate inhibited the oxidation of [U-¹⁴C]glyoxylate to [¹⁴C]oxalate in isolated perfused rat liver, but stimulated total oxalate and glycolate synthesis. [¹⁴C]Oxalate synthesis from [¹⁴C]glycine similarly inhibited by hydroxypyruvate, but conversion of [¹⁴C₁]glycolate to [⁴C]oxalate was increased three-fold. Pyruvate had no effect on the synthesis of [¹⁴C]oxalate or total oxalate. The inhibition studies suggest that hydroxypyruvate is a precursor of glycolate and oxalate and that the conversion of glycolate to oxalate does not involve free glyoxylate as an intermediate. [¹⁴C₃]Hydroxypyruvate, but not [¹⁴C₁]hydroxypyruvate, was oxidized to [¹⁴C]oxalate in isolated perfused rat liver. Isotope dilution studies indicate the major pathway involves the decarboxylation of hydroxypyruvate forming glycolaldehyde which is subsequently oxidized to oxalate via glycolate. The oxidation of serine to oxalate appears to proceed predominantly via hydroxypyruvate rather than glycine or ethanolamine. The hyperoxaluria of L-glyceric aciduria, primary hyperoxaluria type II, is induced by the oxidation of the hydroxypyruvate, which accumulates because of the deficiency of D-glyceric dehydrogenase, to oxalate.     

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.     

Improvement of biomass accumulation of potato plants by transformation of cyanobacterial photorespiratory glycolate catabolism pathway genes

Transgenic potato (Solanum tuberosum L. cv. Desiree) plants expressing components of a novel cyanobacterial photorespiratory glycolate catabolism pathway were developed. Transgenic plant expressing glcD1 (glycolate dehydrogenase I) gene was referred to as synGDH and transgenic plants expressing gcl (glyoxylate carboligase) and tsr (tartronic semialdehyde reductase) genes simultaneously were designated as synGT. Both synGDH and synGT plants showed stable gene transformation, integration and expression. Enhanced glyoxylate contents in synGDH plants were detected as compared to synGT and non-transgenic (NT) plants. Phenotypic evaluation revealed that synGDH plants accumulated 11 % higher dry weight, while, tuber weight was 38 and 16 % higher than NT and synGT, respectively. Upon challenging the plants in high temperature and high light conditions synGDH plants maintained higher Fv/Fm and showed less bleaching of chlorophyll as compared to synGT and NT plants. These results indicate that genetic transformation of complete pathway in one plant holds promising outcomes in terms of biomass accumulation to meet future needs for food and energy.     

A preliminary account of the properties of recombinant human Glyoxylate reductase (GRHPR), LDHA and LDHB with glyoxylate, and their potential roles in its metabolism

Human lactate dehydrogenase (LDH) is thought to contribute to the oxidation of glyoxylate to oxalate and thus to the pathogenesis of disorders of endogenous oxalate overproduction. Glyoxylate reductase (GRHPR) has a potentially protective role metabolising glyoxylate to the less reactive glycolate. In this paper, the kinetic parameters of recombinant human LDHA, LDHB and GR have been compared with respect to their affinity for glyoxylate and related substrates. The Km values and specificity constants (Kcat/K(M)) of purified recombinant human LDHA, LDHB and GRHPR were determined for the reduction of glyoxylate and hydroxypyruvate. K(M) values with glyoxylate were 29.3 mM for LDHA, 9.9 mM for LDHB and 1.0 mM for GRHPR. For the oxidation of glyoxylate, K(M) values were 0.18 mM and 0.26 mM for LDHA and LDHB respectively with NAD+ as cofactor. Overall, under the same reaction conditions, the specificity constants suggest there is a fine balance between the reduction and oxidation reactions of these substrates, suggesting that control is most likely dictated by the ambient concentrations of the respective intracellular cofactors. Neither LDHA nor LDHB utilised glycolate as substrate and NADPH was a poor cofactor with a relative activity less than 3% that of NADH. GRHPR had a higher affinity for NADPH than NADH (K(M) 0.011 mM vs. 2.42 mM). The potential roles of LDH isoforms and GRHPR in oxalate synthesis are discussed.     

Regulation of Glyoxylate Metabolism in Escherichia coli K-12

The relative contributions of the dicarboxylic acid and the tricarboxylic acid cycles to the oxidative catabolism of glyoxylate in Escherichia coli K-12 were deduced by analysis of mutant strains that were blocked in the formation of glyoxylate carboligase and of malate synthase G (the "glycolate form" of malate synthase). Mutant strains unable to form malate synthase G were unimpaired in their ability to oxidize glyoxylate. Hence, the dicarboxylic acid cycle does not appear to play an essential role in this process. Organisms blocked in the synthesis of glyoxylate carboligase did not oxidize glyoxylate at a detectable rate, indicating that wild-type organisms convert glyoxylate to acetyl-coenzyme A and oxidize it via the tricarboxylic acid cycle. The foregoing evidence indicates that malate synthase G plays an anaplerotic role during growth with glycolate or acetate as the carbon source. The in vivo activity of malate synthase G was not detectable when the intracellular concentration of acetyl-coenzyme A was low, suggesting that this substrate or a closely related metabolite exerts a sensitive positive control over the enzyme. The synthesis of malate synthase G appears to be induced directly by glycolate which may be formed by a constitutive reduced nicotinamide adenine dinucleotide phosphate-dependent glyoxylate reductase in glyoxylate- or acetate-grown cells.     

Cytochrome P-450-dependent catabolism of triethanolamine in Rhodotorula mucilaginosa

The yeast Rhodotorula mucilaginosa was able to grow in media containing triethanolamine or diethanolamine as the sole nitrogen source. During growth in the presence of triethanolamine, extracts of yeast cells contained increased levels of cytochrome P-450 dependent monooxygenase which catalyzed the oxidative N-dealkylation of aminoalcohols. Formation of diethanolamine, ethanolamine and glyoxylate from triethanolamine was demonstrated, and the identity of the products was verified by thin layer chromatography. These observations suggested the following scheme of triethanolamine catabolism: triethanolamine diethanolamine + glycolaldehyde, diethanolamine ethanolamine + glycolaldehyde, ethanolamine NH₃ + glycolaldehyde glycolate glyoxylate glycerate pathway.