Presence Of Glyoxylate Research Articles

E. coli allantoinase is activated by the downstream metabolic enzyme, glycerate kinase, and stabilizes the putative allantoin transporter by direct binding

Allantoin is a good source of ammonium for many organisms, and in Escherichia coli it is utilized under anaerobic conditions. We provide evidence that allantoinase (AllB) is allosterically activated by direct binding of the allantoin catabolic enzyme, glycerate 2-kinase (GlxK) in the presence of glyoxylate. Glyoxylate is known to be an effector of the AllR repressor which regulates the allantoin utilization operons in E. coli. AllB has low affinity for allantoin, but its activation by GlxK leads to increased affinity for its substrate. We also show that the predicted allantoin transporter YbbW (re-named AllW) has allantoin specificity and the protein–protein interaction with AllB. Our results show that the AllB-dependent allantoin degradative pathway is subject to previously unrecognized regulatory mechanisms involving direct protein–protein interactions.

Cyanide as a primordial reductant enables a protometabolic reductive glyoxylate pathway.

Investigation of prebiotic metabolic pathways is predominantly based on abiotically replicating the reductive citric acid cycle. While attractive from a parsimony point of view, attempts using metal/mineral-mediated reductions have produced complex mixtures with inefficient and uncontrolled reactions. Here we show that cyanide acts as a mild and efficient reducing agent mediating abiotic transformations of tricarboxylic acid intermediates and derivatives. The hydrolysis of the cyanide adducts followed by their decarboxylation enables the reduction of oxaloacetate to malate and of fumarate to succinate, whereas pyruvate and α-ketoglutarate themselves are not reduced. In the presence of glyoxylate, malonate and malononitrile, alternative pathways emerge that bypass the challenging reductive carboxylation steps to produce metabolic intermediates and compounds found in meteorites. These results suggest a simpler prebiotic forerunner of today's metabolism, involving a reductive glyoxylate pathway without oxaloacetate and α-ketoglutarate-implying that the extant metabolic reductive carboxylation chemistries are an evolutionary invention mediated by complex metalloproteins.

Linked cycles of oxidative decarboxylation of glyoxylate as protometabolic analogs of the citric acid cycle

The development of metabolic approaches towards understanding the origins of life, which have focused mainly on the citric acid (TCA) cycle, have languished—primarily due to a lack of experimentally demonstrable and sustainable cycle(s) of reactions. We show here the existence of a protometabolic analog of the TCA involving two linked cycles, which convert glyoxylate into CO2 and produce aspartic acid in the presence of ammonia. The reactions proceed from either pyruvate, oxaloacetate or malonate in the presence of glyoxylate as the carbon source and hydrogen peroxide as the oxidant under neutral aqueous conditions and at mild temperatures. The reaction pathway demonstrates turnover under controlled conditions. These results indicate that simpler versions of metabolic cycles could have emerged under potential prebiotic conditions, laying the foundation for the appearance of more sophisticated metabolic pathways once control by (polymeric) catalysts became available.

Cyanamide as a prebiotic phosphate activating agent - catalysis by simple 2-oxoacid salts.

Cyanamide is a prebiotically plausible compound that has previously been invoked as a phosphate activating agent. However, its reactions with phosphate monoesters are very slow and tend to be low yielding. We now report a fast and efficient phosphate activation reaction using cyanamide in the presence of glyoxylate or pyruvate. These simple 2-oxoacid salts are shown to function as catalysts and in an optimised system, adenosine-3'-phosphate was converted to adenosine-2',3'-cyclic phosphate in 95% yield.

New insights into the mechanism of substrates trafficking in Glyoxylate/Hydroxypyruvate reductases.

Glyoxylate accumulation within cells is highly toxic. In humans, it is associated with hyperoxaluria type 2 (PH2) leading to renal failure. The glyoxylate content within cells is regulated by the NADPH/NADH dependent glyoxylate/hydroxypyruvate reductases (GRHPR). These are highly conserved enzymes with a dual activity as they are able to reduce glyoxylate to glycolate and to convert hydroxypyruvate into D-glycerate. Despite the determination of high-resolution X-ray structures, the substrate recognition mode of this class of enzymes remains unclear. We determined the structure at 2.0 Å resolution of a thermostable GRHPR from Archaea as a ternary complex in the presence of D-glycerate and NADPH. This shows a binding mode conserved between human and archeal enzymes. We also determined the first structure of GRHPR in presence of glyoxylate at 1.40 Å resolution. This revealed the pivotal role of Leu53 and Trp138 in substrate trafficking. These residues act as gatekeepers at the entrance of a tunnel connecting the active site to protein surface. Taken together, these results allowed us to propose a general model for GRHPR mode of action.

Complete Genome Sequence of Pusillimonas sp. T7-7, a Cold-Tolerant Diesel Oil-Degrading Bacterium Isolated from the Bohai Sea in China

Pusillimonas sp. T7-7 is a diesel oil-degrading cold-tolerant bacterium isolated from the benthal mud of a petroleum-contaminated site in Bohai Sea, China. We present here the complete genome sequence of T7-7. Genome analysis revealed many features of typical marine bacteria, including the absence of intact sugar metabolic pathways, the presence of glyoxylate and gluconeogenesis pathways, and the abilities for nitrate assimilation and denitrification, as well as sulfate reduction and sulfite oxidation. The presence of novel genes for the degradation of diesel oils was suggested.

Biochemical and Structural Studies of Malate Synthase fromMycobacterium tuberculosis

Establishment or maintenance of a persistent infection by Mycobacterium tuberculosis requires the glyoxylate pathway. This is a bypass of the tricarboxylic acid cycle in which isocitrate lyase and malate synthase (GlcB) catalyze the net incorporation of carbon during growth of microorganisms on acetate or fatty acids as the primary carbon source. The glcB gene from M. tuberculosis, which encodes malate synthase, was cloned, and GlcB was expressed in Escherichia coli. The influence of media conditions on expression in M. tuberculosis indicated that this enzyme is regulated differentially to isocitrate lyase. Purified GlcB had K(m) values of 57 and 30 microm for its substrates glyoxylate and acetyl coenzyme A, respectively, and was inhibited by bromopyruvate, oxalate, and phosphoenolpyruvate. The GlcB structure was solved to 2.1-A resolution in the presence of glyoxylate and magnesium. We also report the structure of GlcB in complex with the products of the reaction, coenzyme A and malate, solved to 2.7-A resolution. Coenzyme A binds in a bent conformation, and the details of its interactions are described, together with implications on the enzyme mechanism.

Flow-injection fluorometric quantification of succinate in foodstuffs based on the use of an immobilized enzyme reactor

A method for the quantification of succinate by flow injection analysis was developed using an immobilized-enzyme reactor and a fluorescence detection. Succinate was quantified using a co-immobilized isocitrate lyase (ICL) and isocitrate dehydrogenase (ICDH) reactor. Succinate was converted to isocitrate by ICL in the presence of glyoxylate, and then the produced isocitrate was oxidized with NADP + by ICDH. The NADPH produced by the ICL–ICDH reactor was monitored fluorometrically at 455 nm (excitation at 340 nm). A linear relationship between sensor responses and concentration of succinate was obtained in the range of 5–200 μM. The relative standard deviation for 10 successive injections was 1.01% at the 200 μM level. This analytical method was applied to the quantification of succinate in shellfishes and Japanese sakes, and the results showed good agreement with those obtained using conventional method (F-kit method).

Lactate‐dehydrogenase‐based biosensors for glyoxylate and NADH determination: A novel principle of analyte recyling

AbstractTwo types of bienzyme electrodes based on the oxidation of glyoxylate to oxalate by NAD+ in the presence of lactate dehydrogenase (LDH) are described. The enzymes were entrapped in gelatin membranes and combined with an oxygen probe. A sequence sensor using LDH coupled with oxalate oxidase is useful for the determination of glyoxylate between 1 × 10−5 and 2.5 × 10−3 M. The sensor performance was optimized with respect to pH and cofactor concentration. As a novel approach to internal analyte recycling in biosensors, the LDH‐catalyzed recycling of NADH/NAD+ during the parallel oxidation of glyoxylate and reduction of pyruvate is utilized in a sensor for highly sensitive NADH measurement. Lactate monooxygenase was coupled as an auxiliary enzyme to indicate the lactate formed from pyruvate. The calibration curve of the proposed sensor is linear between 7 × 10−6 and 1.8 × 10−3 M NADH in the absence of glyoxylate (i.e., without analyte recycling) and between 3 × 10−8 and 2 × 10−6 M in the presence of glyoxylate (i.e., with analyte recycling).

Glyoxylate inhibition of ribulosebisphosphate carboxylase/oxygenase activation in intact, lysed, and reconstituted chloroplasts

At bicarbonate concentrations equivalent to air levels of CO2, activation of ribulosebisphosphate carboxylase/oxygenase (rubisco) was inhibited by micromolar concentrations of glyoxylate in intact, lysed, and reconstituted chloroplasts and in stromal extracts. The concentration of glyoxylate required for 50% inhibition of light activation in intact chloroplasts was estimated to be 35 micromolar. No direct inhibition by glyoxylate was observed with purified rubisco or rubisco activase at micromolar concentrations. Levels of ribulose 1,5-bisphosphate and ATP increased in intact chloroplasts following glyoxylate treatment. Results from experiments with well-buffered lysed and reconstituted chloroplast systems ruled out lowering of pH as the cause of inhibition. With intact chloroplasts, micromolar glyoxylate did not prevent activation of rubisco at high (10 mM) concentrations of bicarbonate, indicating that rubisco could be spontaneously activated in the presence of glyoxylate. These results suggest the existence of a component of the in vivo rubisco activation system that is not yet identified and which is inhibited by glyoxylate.

Glycollate-pathway enzymes in mitochondria from phototrophic, organotrophic and mixotrophic cells of Euglena

Glycollate dehydrogenase and NADFH-glyoxylate reductase are constitutive enzymes in Percoll-purified mitochondria from phototrophic, mixotrophic and organotrophic cells of Euglena gracilis Klebs strain z Pringsheim. Glycollate oxidation by isolated mitochondria is stimulated four-fold by the addition of glutamate but rates of glycine oxidation are low in mitochondria from all cell types, the ratio of malate to glycine oxidation always being greater than 4:1. Measurement of the rate of NADPH oxidation in intact mitochondria and mitoplasts showed that the outer mitochondrial membrane is impermeable to NADPH and in the absence of NADPH-dehydrogenase activity the oxidation of NADPH by mitoplasts is dependent on the presence of glyoxylate for NADPH-glyoxylate-reductase activity. It is concluded that glycollate oxidation in the mitochondrion provides glyoxylate which, in the presence of a suitable amino-donor, can be converted to glycine by glutamate-glyoxylate amino-transferase so providing essential intermediates for biosynthesis. Glycollate oxidation outside the mitochondrion is concerned with photorespiratory metabolism and the inability of mitochondria to oxidise exogenous glycine at appreciable rates means that the separation of photorespiratory metabolism from the biosynthesis of essential intermediates is effected.

An enzymatic method for the determination of enantiomeric composition and absolute configuration of deuterated or tritiated succinic acid

The distribution of hydrogen isotope between pro-R and pro-S positions of succinic acid has been determined by comparison of its isotopic content before and after incubation with isocitrate lyase. This enzyme, in the presence of glyoxylate, exchanges exclusively the pro-S protons of succinate with water ( M. Sprecher, R. Berger, and D. B. Sprinson (1964) J. Biol. Chem. 239, 4268–4271). With [1- 14C,2( R,S)- 3H]succinate as substrate, the exchange was easily followed by the decrease of 3H 14 C ratio (dried aliquots), which accounted for the high isotopic effect of this reaction. The final ratio was within ±5% of the theoretical one. The evolution of the exchange of deuterated succinate added with [1- 14C,2( R,S)- 3H]succinic acid was again followed by 3H 14 >C ratio. The deuterium content of [2,3- 2H 2]succinic acid, [2- 2H 2]succinic acid (derived from l-[4- 2H 2]glutamic acid by oxidation) and of the corresponding succinates isolated after incubation with isocitrate lyase was determined by gas chromatography-mass spectroscopy of their dimethylester under NH 4 + chemical ionization. This method provides the basis for a quantitative measurement of the distribution of hydrogen isotopes in unsymmetrically 2-labeled succinate or 4-labeled glutamate.

Stimulation of nitrogenase activity and photosynthesis in some cyanobacteria by glyoxylate

Abstract The effect og glyoxylate on nitrogenase activity (C2H2 reduction) and photosynthesis (H14CO3 fixation and O2 evolution) was in vestigated in the three heterocystous cyanobacteria Anabaena cylindrica, A. variabiltis and N. muscorum. Glyoxylate had virtually no effect on the rate of dark respiration and was unable to sustain photoheterotrophic growth, though some slight stimulation (= 30%) of photorophic growth was noted. A considerable stimulation of both nitrogenase and photosynthetic activities was observed in presence of glyoxylate. In the light the stimulation increased with time up to about 15‐25 h after adding optimal concentrations of 4–6 mM glyoxylate. Placing glyoxylate treated samples in the dark or adding DCMU (30 μM) in the light, showed that glyoxylate initially supported significantly higher nitrogenase activity than did samples in absence of glyoxylate. However, after a prolonged incubation in the dark or in presence of DCMU glyoxylate is unable to relieve the adverse effects of such conditions. The stimulation of the nitrogenase activity was even more pronounced when the glyoxylate was added to cells preincubated in the dark (“carbon starved”) than for cells kept constantly in light. The results suggest that glyoxylate, or a metabolite, may act as an inhibitor of cyanobacterial photorespiration and this hypothesis is discussed.

THE IN VITRO RELEASE OF AMINO ACIDS BY RUMEN PAPILLAE

Rumen papillae from cattle were incubated aerobically with combinations of NH4Cl, amino acids and salts of organic acids, the latter including propionate, pyruvate, α-ketoglutarate and glyoxylate. Amino acids in the incubation media were analyzed using a gas-liquid chromatographic technique entailing separation of the isobutyl-N(0)-heptafluorobutyryl esters: glutamine was recovered with glutamate, asparagine with aspartate, and citrulline with ornithine. Rumen papillae incubated with pyruvate or propionate released alanine, but with the latter substrate only glutamate was effective as a nitrogen source. Glycine and glutamate plus glutamine were released in the presence of glyoxylate and α-ketoglutarate, respectively. Serine and aspartate plus asparagine were not quantitatively major products released by rumen papillae. Glutamate was an effective source of nitrogen for the release of alanine and glycine with pyruvate and glyoxylate, respectively, as carbon sources. When rumen papillae were incubated with pyruvate or glyoxylate as the added carbon source, glutamine nitrogen disappeared and was not accounted for by the amino acids measured. With arginine as a substrate, there was a release of ornithine by rumen papillae indicating urea production. The tissues of rumen papillae appear to synthesize amino acids from expected carbon sources with ammonia or glutamate as nitrogen sources and to catabolize glutamine and arginine. The metabolism of amino acids by rumen papillae would contribute to the interchange of nitrogen between the rumen and the host.

THE IN VITRO SYNTHESIS OF GLYCINE BY RUMEN PAPILLAE

Following aerobic in vitro incubation of papillae from the ventral sac of the bovine rumen, amino acids released into the incubation medium were isolated using a rapid ion exchange procedure and measured quantitatively by gas-liquid chromatographic separation of the isobutyl-N(0)-heptafluorobutyryl esters. Of the potential precursors of the carbon skeleton of glycine that were added as substrates, only glyoxylate and serine (0.5–2 mM) resulted in enhanced glycine release while 10 mM glycolaldehyde, glycollate, glyoxal or oxalate or 1 mM monoethanolamine or hydroxyproline did not specifically influence glycine release. Each increase in glyoxylate concentration through 5, 10 or 20 mM caused an increased glycine release. In the presence of formate, inclusion of glycine as the added substrate increased serine release. Oxalate caused a non-specific release of amino acids. In the presence of glyoxylate, alanine and glutamate (1 mM) were effective precursors of the amino group of glycine.

Effects of Glyoxylate, Glycine, and Serine on the Assimilation of 14CO2 in Mesophyll Cells of Chenopodium album

Mesophyll cells from Chenopodium album were isolated within 1 h in two media containing macerase, hemicellulase, sorbitol, albumine, polyvinylpyrrolidone, potassium dextran sulfate, a mixture of inorganic salts, and MES buffer, pH 5.8. Cells with insufficient activity could be recognized by the absence of phototaxis of their chloroplasts; these cells predominated in preparations from older leaves. The effect of various glycollate metabolites (glycollate, P-glycollate, P-glycolaldehyde, glyoxylate, P-glyoxylate, glycine, and serine) on the assimilation of 14CO2 from H14CO3- at pH 7.3 was investigated. Glyoxylate, glycine, and serine exhibited most pronounced effects depending on the concentration of H14CO3- the total fixation of 14C (control: 34.4 μmoles of CO2/mg of chlorophyll × h) was reduced by 0.01 M solutions of glyoxylate (28.7 μmoles), glycine (23.9 μmoles), and serine (12.9 μmoles). Glyoxylate diminishes the incorporation into amino acids but not into sugars and starch, whereas labelling of sucrose and starch is reduced by glycine and serine. After 1 h of photosynthesis at 5 mM H14CO3- the amino acid fraction above all consists of glutamate and aspartate (67.8 and 13.6%, resp.) followed by alanine (11.0%). The very low radioactivity in serine and glycine (4.0 and 1.2%, resp.) indicates a suppressed glycollate pathway. In 1 mM H14CO3- net photosynthesis is reduced and only little influenced by external glyoxylate, glycine, or serine (control: 8.1 μmoles of CO2/mg of chlorophyll × h; glyoxylate: 7.0; glycine: 8.4; serine: 10.6). Label in glycine (control: 14% of the total amino acids) is reduced in the presence of 0.01 M serine, glycine, or glyoxylate (5.3, 6.3, and 6.7%, resp.). It is suggested that the glycollate pathway might exert a regulatory effect on the net rate of photosynthesis. An increase of net photosynthesis in the presence of glyoxylate could never be observed.

Effect of Glyoxylate on the Sensitivity of Net Photosynthesis to Oxygen (the Warburg Effect) in Tobacco

The addition of glyoxylate to tobacco (Nicotiana tabacum) leaf discs inhibited glycolate synthesis and photorespiration and increased net photosynthetic (14)CO(2) fixation. This inhibition of photorespiration was investigated further by studying the effect of glyoxylate on the stimulation of photosynthesis that occurs when the atmospheric O(2) level was decreased from 21 to 3% (the Warburg effect). The Warburg effect is usually ascribed to the increased glycolate synthesis and metabolism that occurs at higher O(2) concentrations. Photosynthesis in control discs increased from 59.1 to 94.7 micromoles of CO(2) per gram fresh weight per hour (a 60% increase) when the O(2) level was lowered from 21 to 3%, while the rate for discs floated on 15 millimolar glyoxylate increased only from 82.0 to 99.7 micromoles of CO(2) per gram fresh weight per hour (a 22% increase). The decrease in the O(2) sensitivity of photosynthesis in the presence of glyoxylate was explained by changes in the rate of glycolate synthesis under the same conditions.The rate of metabolism of the added glyoxylate by tobacco leaf discs was about 1.35 micromoles per gram fresh weight per hour and was not dependent on the O(2) concentration in the atmosphere. This rate of metabolism is about 10% the amount of stimulation in the rate of CO(2) fixation caused by the glyoxylate treatment on a molar carbon basis. Glyoxylate (10 millimolar) had no effect on the carboxylase/oxygenase activity of isolated ribulose diphosphate carboxylase. Although the biochemical mechanism by which glyoxylate inhibits glycolate synthesis and photorespiration and thereby decreases the Warburg effect is still uncertain, these results show that cellular metabolites can regulate the extent of the Warburg effect.

A Novel Pathway for L-Citramalate Synthesis in Rhodospirillum rubrum1

When [14C]propionate was incubated with a cell-free extract of Rhodospirillum rubrum in the presence of glyoxylate, ATP, CoA, Mg2+, and Mn2+, radioactivity was incorporated into mesaconate (MSA) as well as into beta-methylmalate (MMA) and citramalate (CMA). MSA was suggested to be an intermediate of the conversion of MMA to CMA based on the following observations. (i) When non-labeled MSA was added to the CMA-forming reaction system, radioactivity was trapped in MSA. (ii) When MSA was incubated with the cell-free extract, CMA was formed. (iii) The alpha-carboxyl group of CMA was shown to be derived from the beta-carboxyl group of MMA, implying that CMA was formed from MMA via MSA through successive dehydration and hydration. From the results of Sephadex G-10 column chromatography of the reaction products, beta-methylmalyl-CoA and mesaconyl-CoA were presumed to be involved in the reaction. A new CMA-forming metabolic pathway is proposed as follows: erythro-beta-methylamalyl-CoA leads to mesaconyl-CoA leads to MSA leads to L-CMA.

Properties of methylcitrate synthase from Candida lipolytica.

Methylcitrate condensing enzyme functioning at the entrance to the methylcitric acid cycle was purified 70-fold from cell-free extracts of Candida lipolytica. The purified enzyme preparation catalyzed the liberation of CoASH strongly from propionyl- and acetyl-CoA derivatives and weakly from butyryl- and valeryl-CoA derivatives in the presence of oxaloacetate. The enzyme, however, was specific for oxaloacetate and did not catalyze the liberation of CoASH from acetyl- or propionyl-CoA in the presence of glyoxylate, pyruvate or α-ketoglutarate.All the properties of this preparation were compatible with the conclusion that the sole enzyme, here referred to as methylcitrate synthase, can catalyze the condensation of oxaloacetate not only with propionyl-CoA but also with acetyl-CoA and that this synthase clearly differs from si- and re-citrate synthases.The powerful inhibition of methylcitrate synthase by ATP, NADH and NADPH supports the idea that the physiological role of the methylcitric acid cycle is a cata...

Formation of β-Methylmalate and Its Conversion to Citramalate in Rhodospirillum rubrum

Using a cell-free extract of Rhodospirillum rubrum, studies were made of the condensation reaction between propionyl-CoA and glyoxylate. When [14C]propionate was incubated with the extract in the presence of glyoxylate, ATP, CoA, Mg2+, and Mn2+, radioactivity was incorporated into several compounds. Two of the main products were characterized as citramalate (CMA) and erythro-beta-methylmalate (erythro-MMA) on the basis of their behavior compared with authentic samples of CMA and erythro-MMA in the following three analyses: (i) paper chromatography using two solvent systems, (ii) radio-gas chromatography on their methyl esters, and (iii) chemical conversion to readily crystallizable derivatives, that is, citramalyl chloralide for CMA, and thymine for MMA. The CMA was thought to be of L(+)-form based on the results of optical resolution with brucine and also its susceptibility to L(+)-citramalate lyase of Clostridium tetanomorphum. When the reaction was carried out with lower concentrations of the enzyme, only MMA was accumulated. However, when the reaction was allowed to proceed further after addition of higher concentrations of the enzyme and of excess semicarbazide to prevent further condensation, the amount of accumulated MMA was decreased and CMA was formed instead. Furthermore, the time course of MMA and CMA formation exhibited a pattern typical of a precursor-product relationship. From these results, it was concluded that MMA was formed by alpha-condensation between propionyl-CoA and glyoxylate, and that CMA was derived from MMA, possibly from its CoA derivative.