Oxalate Oxidase Activity Research Articles

A Previously Unknown Oxalyl-CoA Synthetase Is Important for Oxalate Catabolism in Arabidopsis

Oxalate is produced by several catabolic pathways in plants. The best characterized pathway for subsequent oxalate degradation is via oxalate oxidase, but some species, such as Arabidopsis thaliana, have no oxalate oxidase activity. Previously, an alternative pathway was proposed in which oxalyl-CoA synthetase (EC 6.2.1.8) catalyzes the first step, but no gene encoding this function has been found. Here, we identify acyl-activating enzyme3 (AAE3; At3g48990) from Arabidopsis as a gene encoding oxalyl-CoA synthetase. Recombinant AAE3 protein has high activity against oxalate, with K(m) = 149.0 ± 12.7 μM and V(max) = 11.4 ± 1.0 μmol/min/mg protein, but no detectable activity against other organic acids tested. Allelic aae3 mutants lacked oxalyl-CoA synthetase activity and were unable to degrade oxalate into CO(2). Seeds of mutants accumulated oxalate to levels threefold higher than the wild type, resulting in the formation of oxalate crystals. Crystal formation was associated with seed coat defects and substantially reduced germination of mutant seeds. Leaves of mutants were damaged by exogenous oxalate and more susceptible than the wild type to infection by the fungus Sclerotinia sclerotiorum, which produces oxalate as a phytotoxin to aid infection. Our results demonstrate that, in Arabidopsis, oxalyl-CoA synthetase encoded by AAE3 is required for oxalate degradation, for normal seed development, and for defense against an oxalate-producing fungal pathogen.

Oxalic acid and oxalate oxidase enzyme in Costus pictus D. Don

The leaves of Costus pictus are sour in taste due to the presence of oxalic acid in the leaves. Different stages of leaves were collected and the samples were designated as stage one, stage two and stage three. It was found that oxalate content and oxalate oxidase activity were maximum in second leaf stage followed by first leaf stage and third leaf stage. Drying causes substantial loss of oxalate content and complete loss of oxalate oxidase activity. With various solvents water recovered more oxalate followed by methanol and ethanol while oxalate oxidase activity was maximum in ethanol followed by methanol and water. The ethanol or methanol extract of second leaf stage of C. pictus can be used for isolating active principles. The oxalate oxidase from C. pictus can be used as a cheap source of oxalate oxidase enzyme which is used in oxalate determination in biological fluids. Moreover, the sensitivity of oxalate determination employing oxalate oxidase from C. pictus will be more as oxalate oxidase in C. pictus has K m 20 times lesser than the oxalate oxidase enzyme from barley seedling.

Cell wall oxalate oxidase modifies the ferulate metabolism in cell walls of wheat shoots

Oxalate oxidase (OXO) utilizes oxalate to generate hydrogen peroxide, and thereby acts as a source of hydrogen peroxide. The present study was carried out to investigate whether apoplastic OXO modifies the metabolism of cell wall-bound ferulates in wheat seedlings. Histochemical staining of OXO showed that cell walls were strongly stained, indicating the presence of OXO activity in shoot walls. When native cell walls prepared from shoots were incubated with oxalate or hydrogen peroxide, the levels of ester-linked diferulic acid (DFA) isomers were significantly increased. On the other hand, the level of ester-linked ferulic acid (FA) was substantially decreased. The decrease in FA level was accounted neither by the increases in DFA levels nor by the release of FA from cell walls during the incubation. After the extraction of ester-linked ferulates, considerable ultraviolet absorption remained in the hemicellulosic and cellulose fractions, which was increased by the treatment with oxalate or hydrogen peroxide. Therefore, a part of FA esters may form tight linkages within cell wall architecture. These results suggest that cell wall OXO is capable of modifying the metabolism of ester-linked ferulates in cell walls of wheat shoots by promoting the peroxidase action via supply of hydrogen peroxide.

Effects of ionic substances in bleaching filtrates and of lignosulfonates on the activity of oxalate oxidase from barley

The effects of ionic substances in seven industrial filtrates from kraft pulping, mechanical pulping, and sulfite pulping on the activity of oxalate oxidase from barley were investigated by pre-tre ...

Influence of Ca2+ ions on metabolism of active oxygen species in wheat calli cocultured with the bunt pathogen Tilletia caries

The effect of Ca2+ on morphophysiological parameters of wheat calli (Triticum aestivum L.) infected by the bunt pathogen Tilletia caries, in particular on the level of active oxygen species, activity of oxalate oxidase, peroxidase, and catalase is investigated. The concentration of O2−, H2O2, and activity of oxidoreductases (oxalate oxidase, peroxidase, and catalase) depended on the content of Ca2+ in the culture medium of calli. The increase of the concentration of Ca2+ ions in the culture medium led to forming of calli with high structure, induction of activity of oxalate oxidase and of some isoperoxidase, and to accumulation of active oxygen species. These changes contributed to inhibition of development of the fungus. So this dependence confirm the role of calcium as the intermediant in biochemical reactions related to the formation of the protective response of plant cells to biotic stress.

Identification and Characterization of a Multigene Family Encoding Germin-Like Proteins in Cultivated Peanut (Arachis hypogaea L.)

Germin-like proteins (GLPs) play diversified roles in plant development and defense response. Here, we identified 36 expressed sequence tags (ESTs) encoding GLPs from peanut (Arachis hypogaea L.). After assembly, these ESTs were integrated into eight unigenes named AhGLP1 to AhGLP8, of which, three (AhGLP1-3) were comprised 14, ten, and seven EST clones, respectively, whereas the remaining ones were associated with one single clone. The length of the deduced amino acid (AA) residues ranged from 208 to 223 AAs except for AhGLP6 and AhGLP8, which were incomplete at the carboxyl terminus. All of the AhGLPs contained a possible N-terminal signal peptide that was 17 to 24 residues in length excluding AhGLP7, where there is likely a non-cleavable amino terminus. Phylogenetic analysis showed that these AhGLPs were classified into three subfamilies. Southern blot analysis indicated that AhGLP1 and AhGLP2 likely have multiple copies in the peanut genome. The recombinant mature AhGLP1 and AhGLP2 proteins were successfully expressed in Escherichia coli. The purified AhGLP2 has superoxide dismutase (SOD) activity in enzymatic assay, but not oxalate oxidase activity. The SOD activity of AhGLP2 was stable up to 70°C and resistant to hydrogen peroxide, suggesting that AhGLP2 might be a manganese-containing SOD. Furthermore, AhGLP2 could confer E. coli resistance to oxidative damage caused by paraquat, suggesting that the AhGLP2 likely protects peanut plants from reactive oxygen metabolites. Thus, information provided in this study indicates the diverse nature of the peanut GLP family and suggests that some of AhGLPs might be involved in plant defense response.

Strawberry Fruit Oxalate Oxidase — Detection, Purification, Characterization and Physiological Role

A soluble oxalate oxidase activity has been detected in homogenate of ripened fruits of strawberry (Fragaria ananassa), as confirmed by the stoichiometric relationship between the disappearance of oxalate and utilization of dissolved O2, and generation of H2O2. The enzyme was purified up to apparent homogeneity and had a Mr of 119 kDa with two identical subunits. Km for oxalate was found to be 1.67×10−3 M, and Vmax of 0.741 mmoles ml−1min−1. It retained 76% of its initial activity, when heated at 60°C for 30 min. The enzyme was found to be glycoprotein in nature. The significant increase in the enzyme activity of ripened fruits compared to that in pre-ripened fruit, and decrease in oxalate level (−0.927 correlation with oxalate oxidase) with advancement of ripening indicated the physiological role of enzyme in fruit ripening.

Effect of lectins from Azospirillum brasilense to peroxidase and oxalate oxidase activity regulation in wheat roots

Lectins were extracted from the surface of nitrogen-fixing soil bacteria Azospirillum brasilense Sp7 and from its mutant A. brasilense Sp7.2.3 defective in lectin activity. The ability oflectins to stimulate the rapid formation of hydrogen peroxide related to increase of oxalate oxidase and peroxidase activity in the roots of wheat seedlings has been demonstrated. The most rapid induced pathway of hydrogen peroxide formation in the roots of wheat seedlings was the oxalic acid oxidation by oxalate oxidase which is the effect oflectin in under 10 min in a concentration of 10 microg/ml. The obtained results show that lectins from Azospirillum are capable of inducing the adaptation processes in the roots of wheat seedlings.

The Effects of Oxyanions on the Activity of Oxalate Oxidase

Oxalate oxidase catalyzes the conversion of oxalic acid and molecular oxygen to carbon dioxide and hydrogen peroxide. Oxalate-degrading enzymes are of interest for various applications including clinical analysis of the levels of oxalic acid in blood and urine and control of oxalic acid in industrial processes, such as pulp and paper manufacture. In these applications, the presence of oxyanions other than oxalate may affect the enzyme activity. The inhibitory effect of selected oxyanions on oxalate oxidase from barley was investigated. Seven out of fourteen of the compounds studied inhibited oxalate oxidase at a concentration of 1 mM. Perchlorate, chlorate and chlorite were selected for more detailed studies. The results indicate that perchlorate, chlorate and chlorite cause mixed inhibition of oxalate oxidase and that the severity of the inhibition within the series increases with the oxidation state. The apparent KM of the enzyme was 0.28 ± 0.05 mM.

Role of reactive oxygen species-generating enzymes and hydrogen peroxide during cadmium, mercury and osmotic stresses in barley root tip

The effect of cadmium (Cd) on the expression and activity of NADPH oxidase, peroxidase and oxalate oxidase as well as on the expression of aquaporins and dehydrins was studied in barley root tip. The root tip represented intact apical part of the barley root containing the root cap, meristems and elongation zone. Except stress induced by Cd, barley root tips were analysed after their exposure to phytotoxic concentration of mercury (Hg)-, hydrogen peroxide (H2O2)- or polyethylene glycol (PEG)-induced water stress in order to compare the Cd-induced changes with changes induced by these other stress factors. Cd, Hg, H2O2 and with some exceptions also PEG treatments caused similar alterations in the gene expression of reactive oxygen species (ROS)-generating and water deficiency-related genes, and in the activity of ROS-generating enzymes. These evidences support our opinion that ROS accumulation and water imbalance are the common symptoms of these stress factors and that the elevated production of H2O2 plays, probably as a signal molecule, a key role in the induction of plant responses to abiotic stresses in barley root tip. On the other hand, H2O2 at permanent high concentration is probably the main toxic factor during stress conditions.

Hydrogen peroxide production in wheat leaves infected with the fungus Septoria nodorum Berk. Strains with different virulence

The effect of two strains of the phytopathogenic fungus Septoria nodorum Berk. of different virulence on the intensity of local generation of hydrogen peroxide in common wheat leaves and the role of oxidoreductases in this process was studied. Differences in the pattern of hydrogen peroxide production in wheat plants infected with high- and low-virulence pathogen strains have been found. The low-virulent S. nodorum strain caused a long-term hydrogen peroxide (H2O2) generation in the infection zone, whereas the inoculation of leaves with the highly virulent strain resulted in a transient short-term increase in the H2O2 concentration at the initial moment of contact between the plant and the fungus. It was shown that the low level of H2O2 prOduction by plant cells at the initial stages of pathogenesis facilitates S. nodorum growth and development. The decrease in the H2O2 concentration induced by the highly virulent S. nodorum strain is determined by inhibition of the oxalate oxidase activity in plant tissues and by the ability of the fungus to actively synthesize an extracellular catalase. The pattern of hydrogen peroxide generation at the initial stages of septoriosis may serve as an index of virulence of S. nodorum population.

Increased tolerance to oxidative stress in transgenic tobacco expressing a wheat oxalate oxidase gene via induction of antioxidant enzymes is mediated by H2O2

Hydrogen peroxide (H(2)O(2)) plays a key role in the regulation of plant responses to various environmental stresses and modulates the expression of related genes including those encoding antioxidant enzymes. A wheat oxalate oxidase (OxO) gene was transformed and expressed in tobacco for production of H(2)O(2). The transgenic plants exhibited enhanced OxO activities and H(2)O(2) concentrations, which was blocked by inhibitors of OxO. The transgenic plants showed increased tolerance to methyl viologen (MV) or high light-induced oxidative stress in both short-time and long-time tests by measuring their maximal photochemical efficiency of PSII (F(v)/F(m)), ion leakage and malondialdehyde. Higher activities and transcripts of antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase) were observed in the transgenic plants compared to their wild-type controls under normal growth conditions. Pretreatments with inhibitors of OxO and scavenger of H(2)O(2) blocked the increase of tolerance to MV-induced or high light-induced oxidative stress, as well as the induction of antioxidant enzyme activities. Pretreatments with H(2)O(2) increased tolerance to oxidative stresses and antioxidant enzyme activities. It is suggested that H(2)O(2) produced by OxO in the transgenic tobacco plants triggers the signaling pathways to upregulate expressions of antioxidant enzyme genes, which in turn results in the increase of tolerance to MV-induced and high light-induced oxidative stresses.

Increase of ascorbic acid content and nutritional quality in spinach leaves during physiological acclimation to low temperature

The effect of acclimation to 10 degrees C on the leaf content of ascorbic and oxalic acids, was investigated in spinach (Spinacia oleracea L.). At 10 degrees C the content of ascorbic acid in leaves increased and after 7 days it was about 41% higher than in plants remaining under a 25 degrees C/20 degrees C day/night temperature regime. In contrast, the content of oxalate, remained unchanged. Transfer to 10 degrees C increased the ascorbic but not the oxalic acid content of the leaf intercellular washing fluid (IWF). Oxalate oxidase (OXO EC 1.2.3.4) activity was not detected in extracts of leaf blades. Therefore, oxalic acid degradation via OXO was not involved in the control of its content. Our results show that low temperature acclimation increases nutritional quality of spinach leaves via a physiological rise of ascorbic acid that does not feed-forward on the content of oxalic acid.

Effect of heavy metals and temperature on the oxalate oxidase activity and lignification of metaxylem vessels in barley roots

The effect of Cd on oxalate oxidase (OxO) activity and its localisation were analysed in barley root. In Cd-treated roots OxO activity was strongly induced in the region 2–4 mm behind the root tip and in the area toward the root base. In situ analyses showed that Cd-induced OxO activity was localised to the cell wall (CW) of early metaxylem vascular bundles and surrounding parenchyma cells and was accompanied by lignification of metaxylem vessels. OxO activation was also observed during treatment with other heavy metals (HMs), salt treatment and at elevated non-optimal temperature. In contrast to HM activation of OxO and lignification, high temperature and NaCl indeed activated OxO but did not induce lignification of metaxylem vessels. These results suggest that oxalate oxidase as an H 2O 2-generating enzyme is activated in response to several stresses, however the ectopic lignification of metaxylem vessels is activated specifically by HMs. This HM-induced premature root xylogenesis due to ectopic lignification of metaxylem vessels probably causes shortening of the root elongation zone and therefore a reduction in root growth.

Abortiporus biennis tolerance to insoluble metal oxides: oxalate secretion, oxalate oxidase activity, and mycelial morphology

The ability of Abortiporus biennis to tolerate and solubilize toxic metal oxides (Cu(2)O, Al(2)O(3), ZnO, CuFe(2)O(4)Zn, CdO, and MnO(2)) incorporated into agar media was investigated and the growth rate, oxalic acid secretion, and mycelial morphology were monitored. Among the tested metal oxides, formation of clear zones underneath the mycelium growing on Cu(2)O- and ZnO-amended plates was observed. ZnO, CdO and Cu(2)O caused the highest rate of fungal growth inhibition. An increased level of oxalic acid concentration was detected as a response of A. biennis to the presence of Cu(2)O, MnO(2), ZnO and CuFe(2)O(4)Zn in growth medium. The oxalate oxidase (OXO) was found to be responsible for oxalic acid degradation in A. biennis cultivated in metal-amended media. An increased level of OXO was observed in media amended with Cu(2)O, ZnO and MnO(2). Confocal microscopy used in this study revealed changes in mycelial morphology which appeared as increased hyphal branching, increased septation and increased spore number.

Effect of hydrogen peroxide on morphological characteristics and resistance of wheat calluses to T. caries Tul.

We studied the effect of hydrogen peroxide on morphological characteristics and resistance of common wheat calluses ( Triticum aestivum L.) to Tilletia caries Tul. The induction of the defense response and morphogenesis in calluses depended on H2O2 concentration. A correlation was revealed between the elevated concentration of hydrogen peroxide in wheat calluses and high activity of oxalate oxidase in the cell wall. Administration of H2O2 into the callus culture medium was followed by rhizogenesis, induced the formation of dense regions, and inhibited fungal growth on calluses. Hydrogen peroxide at high concentrations was less potent in inhibiting the growth of fungi. A relationship was found between oxalate oxidase activity, H2O2 concentration, and morphogenetic and defense responses of calluses induced by exogenous hydrogen peroxide. These data suggest that the induction of H2O2 generation is one of the approaches to increase callus resistance.

Expressing a gene encoding wheat oxalate oxidase enhances resistance to Sclerotinia sclerotiorum in oilseed rape (Brassica napus)

Sclerotinia sclerotiorum causes a highly destructive disease in oilseed rape (Brassica napus). Oxalic acid (OA) secreted by the pathogen is a key pathogenicity factor. Oxalate oxidase (OXO) can oxidize OA into CO2 and H2O2. In this study, we show that transgenic oilseed rape (sixth generation lines) constitutively expressing wheat (Triticum aestivum) OXO displays considerably increased OXO activity and enhanced resistance to S. sclerotiorum (with up to 90.2 and 88.4% disease reductions compared with the untransformed parent line and a resistant control, respectively). Upon application of exogenous OA, the pH values in transgenic plants were maintained at levels slightly lower than 5.58 measured prior to OA treatment, whereas the pH values in untransformed plants decreased rapidly and were markedly lower than 5.63 measured prior to OA treatment. Following pathogen inoculation, H2O2 levels were higher in transgenic plants than in untransformed plants. These results indicate that the enhanced resistance of the OXO transgenic oilseed rape to Sclerotinia is probably mediated by OA detoxification. We believe that enhancing the OA metabolism of oilseed rape in this way will be an effective strategy for improving resistance to S. sclerotiorum.

Expression of an oxalate oxidase gene in tomato and severity of disease caused by Botrytis cinerea and Sclerotinia sclerotiorum

A cDNA clone of wheat oxalate oxidase (OxO) under the control of the constitutive CAMV 35S promotor was expressed in tomato plants by Agrobacterium‐mediated transformation. Twenty‐six transgenic tomato lines were obtained and analysed. PCR experiments confirmed the incorporation of the OxO gene in all tested tomato lines. The transgenic tomato plants expressed a 124‐kDa protein showing OxO activity, and were able to convert different oxalic acid (OA) concentrations in vitro. In a detached leaf assay, most of the transgenic lines showed reduced disease symptoms compared with controls, following inoculation with Botrytis cinerea. In addition, leaves of the line T15 showed a marked reduction in symptoms compared with the control following inoculation with Sclerotinia sclerotiorum.

Oxalate oxidase and non-enzymatic compounds of the antioxidative system in young Serbian spruce plants exposed to cadmium stress

We studied changes in the concentrations of ascorbate and glutathione, composition of soluble phenolics, and activity of oxalate oxidase in 75-day-old Serbian spruce plants after exposure to 5 ?M and 50?M cadmium for 6-48 h. The presence of OxOxactivity in a conifer species is here demonstrated for the first time. Both Cd concentrations induced a decrease of OxOxactivity in treated plants in comparison with the control at all sampling dates. The concentrations of reduced glutathione, its oxidized form, and reduced ascorbate in the plants decreased during 48-h treatment with cadmium. Among simple phenolics, only catechin increased significantly during Cd treatment.

Enzyme-based control of oxalic acid in the pulp and paper industry

Enzymatically catalyzed decomposition of oxalic acid in bleaching filtrates from the pulp and paper industry offers a possibility to enduringly prevent oxalate scaling problems by specific removal of the oxalic acid in the system rather than by attempting to avoid calcium oxalate precipitation by countermeasures aiming at improved solubility. To achieve a broad evaluation of various oxalate-degrading enzymes and to cover conditions encountered in various types of processes, 16 different bleaching filtrates were collected from pulp mills engaged in mechanical pulping of softwood, mechanical pulping of aspen, and kraft pulping of softwood. A novel oxalate-degrading enzyme provided by Novozymes was compared with commercially available oxalate oxidase from barley and oxalate decarboxylase from Aspergillus niger. The activity of the enzymes in the filtrates was investigated using kinetic analysis and multivariate data analysis. Kinetic analysis indicated that the degradation rates were governed more by inhibitors in the filtrates than by the concentration of oxalic acid. Multivariate data analysis suggested links between high concentrations of certain compounds in the filtrates and high or low enzyme activity, as exemplified by the link between high concentrations of chelators in filtrates from mechanical pulping and low activity of oxalate oxidase from barley. All three enzymes could degrade oxalic acid in all filtrates, despite the fact that very high concentrations of residual hydrogen peroxide were found in several of the filtrates.