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We have described for the first time the localisation of oxalate oxidase (OXO, EC 1.2.3.4) in Abortiporus biennis cells, using histochemical and immunochemical methods coupled with transmission electron microscopy. Rabbit anti-oxalate oxidase immunoglobulins with anti-rabbit secondary antibody conjugated with 10-nm gold particles were used. Moreover, the formation of electron dense precipitation of reaction of diaminobenzidine (DAB) with horseradish peroxidase (HRP) for histochemical localisation of the enzyme was found. OXO was localised close to the membranous structures of the cell membranes, in membranous vesicles located close to the outer cell membrane, and vacuolar membranes surrounding vacuoles. The positive immunoreaction to OXO was also intense in cell wall areas. Moreover, we proved that gene coding for OXO was expressed in the same cultures. Corresponding mRNA was isolated, full length cDNA was synthesized, cloned and sequenced. Two copies of cupin domains were found in the sequence of amino-acids conserved domain coding for the cupin enzyme. Comparison of the genomic DNA and cDNA sequences has revealed the presence of seventeen introns in the gene. The isoelectric point of the protein was estimated at pH 4.5 and several possible N-glycosylation sites were predicted.

Over the past few years, three photorespiratory bypasses have been introduced into plants, two of which led to observable increases in photosynthesis and biomass yield. However, most of the experiments were carried out using Arabidopsis under controlled environmental conditions, and the increases were only observed under low-light and short-day conditions. In this study, we designed a new photorespiratory bypass (called GOC bypass), characterized by no reducing equivalents being produced during a complete oxidation of glycolate into CO2 catalyzed by three rice-self-originating enzymes, i.e., glycolate oxidase, oxalate oxidase, and catalase. We successfully established this bypass in rice chloroplasts using a multi-gene assembly and transformation system. Transgenic rice plants carrying GOC bypass (GOC plants) showed significant increases in photosynthesis efficiency, biomass yield, and nitrogen content, as well as several other CO2-enriched phenotypes under both greenhouse and field conditions. Grain yield of GOC plants varied depending on seeding season and was increased significantly in the spring. We further demonstrated that GOC plants had significant advantages under high-light conditions and that the improvements in GOC plants resulted primarily from a photosynthetic CO2-concentrating effect rather than from improved energy balance. Taken together, our results reveal that engineering a newly designed chloroplastic photorespiratory bypass could increase photosynthetic efficiency and yield of rice plants grown in field conditions, particularly under high light.

Germins and germin-like proteins (GLPs) are ubiquitious glycoproteins in the plant kingdom. They play critical roles in both plant development and responses to stresses. Here we report the identification and functional characterization of the GLP gene family in tea plant (Camellia sinensis). By mining the leaf transcriptomes of tea plants, eight genes encoding GLPs were identified, they were designated as CsGLP1-8. Phylogenetic analysis placed the eight CsGLPs into three subfamilies, with CsGLP4 and CsGLP3 in the subfamily I and II, respectively and all the rest in subfamily III. The functions of CsGLPs were further characterized at both biochemical and biological levels. Analysis of recombinant proteins produced by expressing full-length cDNAs of individual CsGLPs in E. coli revealed that all CsGLPs had oxalate oxidase (OxO) or superoxide dismutase (SOD) activities, which are considered as typical biochemical characteristics of GLPs. In addition, expression analysis of individual CsGLP genes during bud sprouting as well as under biotic and abiotic stresses further confirmed their involvements in tea plant development and stress response. In apical buds, CsGLP genes exhibited differential response to phenological changes. CsGLP2 increased more extremely at bud sprouting stage than dormant stage compared with other genes, signifying its potentiality as a bud germination marker for the selection of germplasms in tea breeding. Under drought and H2O2 stresses, the expression levels of CsGLP almost constantly down-regulated, suggesting that bud sprouting was suppressed by water shortage and oxidative stress. For biotic treatment, six genes showed obvious positive response to herbivore-induced damage. Especially, CsGLP1 and CsGLP2 were dramatically induced by geometrid feeding, indicating that these two GLP members may be involved in insect resistance in tea plant. The results of this study will help to better understand the function of GLPs family in tea plants and to further exploit this knowledge for tea breeding.

Oxalic acid, the simplest of all dicarboxylic acids, is widely found in all plant species (Franceschi and Nakata, 2005). Two pathways exist for oxalate degradation in plants: degradation via oxalate oxidase and ATP- and CoA-dependent decarboxylation of oxalate through a poorly studied series of enzymatic reactions (Giovanelli and Tobin, 1961). Oxalyl-CoA synthetase, the enzyme that initiates the second degradation pathway was recently identified in Arabidopsis thaliana as a protein encoded by ACYL-ACTIVATING ENZYME3 (AAE3).

Isothermal titration calorimetry (ITC) may be used to determine the kinetic parameters of enzyme-catalyzed reactions when neither products nor reactants are spectrophotometrically visible and when the reaction products are unknown. We report here the use of the multiple injection method of ITC to characterize the catalytic properties of oxalate oxidase (OxOx) from Ceriporiopsis subvermispora (CsOxOx), a manganese dependent enzyme that catalyzes the oxygen-dependent oxidation of oxalate to carbon dioxide in a reaction coupled with the formation of hydrogen peroxide. CsOxOx is the first bicupin enzyme identified that catalyzes this reaction. The multiple injection ITC method of measuring OxOx activity involves continuous, real-time detection of the amount of heat generated (dQ) during catalysis, which is equal to the number of moles of product produced times the enthalpy of the reaction (ΔHapp). Steady-state kinetic constants using oxalate as the substrate determined by multiple injection ITC are comparable to those obtained by a continuous spectrophotometric assay in which H2O2 production is coupled to the horseradish peroxidase-catalyzed oxidation of 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) and by membrane inlet mass spectrometry. Additionally, we used multiple injection ITC to identify mesoxalate as a substrate for the CsOxOx-catalyzed reaction, with a kinetic parameters comparable to that of oxalate, and to identify a number of small molecule carboxylic acid compounds that also serve as substrates for the enzyme.

Four oxalate oxidase (OxO) positive fungal strains were isolated from rotting taro corms. One isolate showing highest extra cellular OxO titre was selected and was identified based on partial sequencing of 18S rRNA. The selected strain was found to be Fusarium oxysporum f. sp. cumini. Oxalate oxidase (EC 1.2.3.4) titre was increased by one factor at a time approach in shake flasks. The enzyme was then purified by isopropanol precipitation followed by chromatography using ENrich SEC650 FPLC column. OxO was found to be a heterodimer having two subunits of mass 116 and 105 kDa. Optimum pH and temperature were found to be 3.8 and 80 °C respectively. OxO exhibited stability for 4 h at temperature range of 4–95 °C and pH between 2.2 and 10.0. OxO was inactive until 30 °C and more than 90% of its peak activity was observed at temperature above 80 °C, suggesting that the enzyme is thermophilic. OxO appears to be a metalloprotein inhibited by Cu2+, Fe2+ and EDTA and enhanced in the presence of Mn2+ and biotin. OxO also exhibited broad substrate specificity. This enzyme was found to decompose insoluble calcium oxalate crystals. This is the first report of OxO decomposing calcium oxalate crystals.

The biochemical and enzymatic properties of four highly similar rice oxalate oxidase proteins (OsOxO1-4) were compared after their purification from the leaves of transgenic plants each overexpressing the respective OsOxO1-4 genes. Although alignment of their amino acid sequences has revealed divergence mainly in the signal peptides and they catalyze the same enzymic (oxalate oxidase) reaction, divergence in apparent molecular mass, Km, optimum pH, stability and responses to inhibitors and activators was uncovered by biochemical characterization of the purified OsOxO1-4 proteins. The apparent molecular mass of oligomer OsOxO1 was found to be similar to that of OsOxO3 but lower than the other two. The molecular mass of the subunit of OsOxO1 was lower than that of OsOxO3. The Km value of OsOxO3 was higher than the other three which had similar Km. OsOxO1 and OsOxO4 possessed peak activity at pH 8.5 which was close to that at the optimum pH 4.0. The activity of OsOxO2 at pH 8.5 was only 65% of that at its optimum pH 3.5, while the activity of OsOxO3 did not vary much at pH 6-9 and was also much lower than that at its optimum pH 3. OsOxO2 and OsOxO3 still maintained all their activities after being heated at 70°C for 1h while OsOxO1 and OsOxO4 lost about 30% of their activities. Pyruvate and oxaloacetic acid inhibited the activity of OsOxO3 more strongly than the other three. Interestingly, glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-biphosphate related to photosynthetic assimilation of triose phosphate greatly increased the activities of OsOxO3 and OsOxO4. In addition to the differences in the biochemical properties of the four OsOxO proteins, an intriguing finding is that the purified OsOxO1-4 exhibited substrate inhibition, which is a typical of the classical Michaelis-Menten enzyme kinetics exhibited by a majority of other enzymes.

Multi-year transgenic field trials were conducted to assess the extent of pollen-mediated transgenic flow in Virginia to support a petition requesting deregulated status for Blight Blocker peanuts from USDA Animal and Plant Health Inspection Service (APHIS) Biotechnology Regulatory Services (BRS). We measured transgene flow from transgenic lines to their non-transgenic parental cultivars. A colorimetric method based on quantification of hydrogen peroxide released from oxalic acid in the presence of the oxalate oxidase was used to screen seed embryos from non-transgenic rows at various distances from the transgenic source. The overall transgene flow rate in three cultivars was 0.2094% based on screening over 85,000 seeds. In general, the transgene flow rate greatly declined past 10 m from the transgene source. However, a transgene flow rate of less than 0.05% did occur sporadically at greater distances than 10 m. In conclusion, transgene flow in peanut can be spatially confined to provide negligible rates using relatively short separation distances. The extremely low rate of transgene flow at greater distance was dependent on ecological and environmental contexts, particularly on foraging patterns and flight distance of pollinators.

Membrane inlet mass spectrometry (MIMS) uses a semipermeable membrane as an inlet to a mass spectrometer for the measurement of the concentration of small uncharged molecules in solution. We report the use of MIMS to characterize the catalytic properties of oxalate oxidase (E.C. 1.2.3.4) from Ceriporiopsis subvermispora (CsOxOx). Oxalate oxidase is a manganese dependent enzyme that catalyzes the oxygen-dependent oxidation of oxalate to carbon dioxide in a reaction that is coupled with the formation of hydrogen peroxide. CsOxOx is the first bicupin enzyme identified that catalyzes this reaction. The MIMS method of measuring OxOx activity involves continuous, real-time direct detection of oxygen consumption and carbon dioxide production from the ion currents of their respective mass peaks. 13 C 2 -oxalate was used to allow for accurate detection of 13 CO 2 ( m / z 45) despite the presence of adventitious 12 CO 2 . Steady-state kinetic constants determined by MIMS are comparable to those obtained by a continuous spectrophotometric assay in which H 2 O 2 production is coupled to the horseradish peroxidase catalyzed oxidation of 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid). Furthermore, we used MIMS to determine that NO inhibits the activity of the CsOxOx with a K I of 0.58 ± 0.06 μM.

Abscisic acid (ABA), H2O2 and nitric oxide (NO) are important signals in gene expression and physiological responses during plant adaptation to environmental stresses. The essential role of NR-derived NO production in ABA and H2O2 induced antioxidant enzymes were studied using transgenic tobacco plants over-expressing Stylosanthes guianensis 9-cis-epoxycartenoid dioxygenase gene (SgNCED1) for elevated ABA level, or over-expressing wheat oxalate oxidase gene (OxO) for elevated H2O2 level in comparison to the wild type. Compared to the wild type, higher levels of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and nitrate reductase (NR) activities and NO production were observed in all transgenic plants. For investigating the relationship of ABA, H2O2, and NR-produced NO in the induction of antioxidant enzyme activities, an inhibitor of ABA biosynthesis, scavengers of H2O2 and NO, and an inhibitor of NR were used in the experiments. The results indicate that H2O2-induced activities of SOD, CAT, and APX depends on NR-derived NO in OxO transgenic plants, while ABA-induced activities depends on H2O2 and NR-derived NO in SgNCED1 transgenic plants. Compared to unaltered nitrate reductase 2 (NIA2), NIA1 transcript was induced in both types of transgenic plants. It is suggested NR-derived NO is essential for ABA- or H2O2-induced antioxidant enzyme activities.

The total oxalate contents of various vegetables, legume seeds, cereals, fruits and nuts commonly available in Southern China were determined using an improved enzymatic method. Spinach, xeric water spinach (grows on moist soil), amaranth, bamboo shoot, ginger, Chinese wolfberry, rice bean and black glutinous rice contained more than 100mg oxalate/100gFW, but leaf mustard, white radish, broccoli, cauliflower, cabbage, onion, gourd except bitter melon, tomato, pepper, chufa, kelp, mushroom, soybean sprout and cowpea contained less than 10mg oxalate/100gFW. The oxalate contents of star fruit and dragon fruit were 111.4 and 97.1mg/100gFW, respectively, and those of other fruits except strawberry and black plum were less than 20mg/100gFW. Almond, cashew, hazel, pine nut and abalone fruit contained more than 150mg total oxalate/100gFW, and the oxalate levels in Chinese torreya fruit, peanut, pistachio and walnut ranged from 54.1 to 83.1mg/100gFW. Based on the results obtained, water spinach, Chinese wolfberry, black glutinous rice, dragon fruit, rice bean, abalone fruit and Chinese torreya fruit should also be considered as high oxalate-foods. The enzymatic method used in the present study enabled us to perform a large-scale quantitative investigation of oxalate contents of different foods.

Oxalate oxidase has potential to act as an oxygen scavenger in active packaging to increase the shelf-life of food and beverages, while simultaneously producing the protective packaging gas carbon ...

Seven bacterial isolates screened from rhizosphere of common bean growing at Uttarakhand Himalaya showed potential plant growth promoting (PGP) and antagonistic activities. Based on 16S rRNA gene sequence the isolate BPR7 was identified as Bacillus sp. BPR7. The strain BPR7 produced IAA, siderophore, phytase, organic acid, ACC deaminase, cyanogens, lytic enzymes, oxalate oxidase, and solubilized various sources of organic and inorganic phosphates as well as potassium and zinc. Strain BPR7 strongly inhibited the growth of several phytopathogens such as Macrophomina phaseolina, Fusarium oxysporum, F. solani, Sclerotinia sclerotiorum, Rhizoctonia solani and Colletotricum sp. in vitro. Cell-free culture filtrate of strain BPR7 also caused colony growth inhibition of all test pathogens. PGP and antifungal activities of Bacillus sp. BPR7 suggest that it may be exploited as a potential bioinoculant agent for P. vulgaris.

Objectives The use of Au nanoparticles (AuNPs)–CaCO3 porous microsphere hybrid as a matrix for enzyme immobilization to increase the performance of an amperometric biosensor. Design and methods A method is described for construction of an amperometric oxalate biosensor by immobilizing a barley root oxalate oxidase (OxOx) onto AuNPs–CaCO3 porous microsphere hybrid encapsulated in silica sol and deposited on Au electrode. Results The biosensor showed optimum response within 4 s at pH 5.0 and 30 °C, when polarized at + 0.4 V vs. Ag/agCl. The biosensor possesses high sensitivity and measures oxalate concentrations as low as 1.0 μmol/L. The working linear range was from 1.0 to 1000 μmol/L of oxalate. The biosensor measured urinary and plasma oxalate of normal persons and stone formers. Conclusion The use of AuNPs–CaCO3 porous microsphere hybrid as a support for immobilization of OxOx has resulted into an improved amperometric oxalate biosensor.

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.

An amperometric oxalate biosensor using nanohybrid film of multi-walled carbon nanotubes (MWCNTs) and gold colloidal nanoparticles (GNPs) via carbodiimide chemistry by forming amide linkages between carboxylic acid groups on the CNTs and amine residues of cysteamine self-assembled monolayer (SAM) has been prepared. The c-MWCNTs were immobilized on the gold (Au) electrode and characterized by FTIR. The morphologies of the c-MWCNT/Au and GNPs/MWCNT/Au electrodes were investigated by scanning electron microscopy (SEM) and the electrochemical performance of the Au, c-MWCNT/Au and GNPs/c-MWCNT/Au electrodes were also studied amperometrically. The Cl− and NO3− insensitive oxalate oxidase from grain sorghum was finally immobilized on this electrode. The influence of pH, temperature and oxalate concentration on electrode activity was studied. The electrode showed optimum response within 7 s. The electrocatalytic response showed a linear dependence on the oxalic acid concentration ranging from 1 to 800 μM with a detection limit of 1 μM. The Km value for the oxalic acid sensor was 444.44 μM. The enzyme electrode retained 30% of its initial activity after 5 months, when stored at 4 °C. The electrode was employed for measurement of oxalic acid in serum, urine and foodstuffs.

Oxalate oxidase (E.C. 1.2.3.4) catalyzes the oxygen-dependent oxidation of oxalate to carbon dioxide in a reaction that is coupled with the formation of hydrogen peroxide. Although there is currently no structural information available for oxalate oxidase from Ceriporiopsis subvermispora (CsOxOx), sequence data and homology modeling indicate that it is the first manganese-containing bicupin enzyme identified that catalyzes this reaction. Interestingly, CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC). We show that CsOxOx activity directly correlates with Mn content and other metals do not appear to be able to support catalysis. EPR spectra indicate that the Mn is present as Mn(II), and are consistent with the coordination environment expected from homology modeling with known X-ray crystal structures of OxDC from Bacillus subtilis. EPR spin-trapping experiments support the existence of an oxalate-derived radical species formed during turnover. Acetate and a number of other small molecule carboxylic acids are competitive inhibitors for oxalate in the CsOxOx catalyzed reaction. The pH dependence of this reaction suggests that the dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be unprotonated.

Glycerol has drawn increasing attention as a possible fuel, because it has many desirable qualities and is abundant due to the fact that it is a byproduct of biodiesel production. Previous research has shown that non-natural enzyme cascades can be used to create a bioanode that can stepwise oxidize glycerol to carbon dioxide. Two of these enzymes are pyrroloquinoline quinone (PQQ) dependant alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AldDH) derived from Gluconobacter. The third enzyme, which is responsible for carbon bond cleavage, is oxalate oxidase (OxOx) derived from barley. Previous research has shown that all three enzymes have demonstrated the ability to undergo direct electron transfer to a carbon electrode which allows for a simple and efficient bioanode that completely oxidizes glycerol. In this study, each enzyme was individually immobilized within modified Nafion ® on a glassy carbon rotating disc electrode (GC-RDE) and voltammetric analysis was performed employing different rotation rates in a solution containing each enzyme's respective substrate. This substrate was glycerol for alcohol dehydrogenase, glyceraldehyde for aldehyde dehydrogenase, and mesoxalic acid for oxalate oxidase. From the voltammograms, Levich plots were produced and the solution diffusion coefficient ( D soln ), the membrane diffusion coefficient ( D film ), k CAT , K M , and V MAX were determined.

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 2 O 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.

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.

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.

Activated polystyrene cuvet is used for the determination of oxalate in urine sample and immunoglobulin in human sera. Polystyrene cuvet was activated by introducing active functional group onto its surface by coating the surface with a heterobifunctional photolinker, 1-fluoro-2-nitro-4-azidobenzene and exposing to UV light at 365 nm. Modified cuvet was active enough to covalently link oxalate oxidase in just 45 min at 50 °C without any additional catalyst or reagent. Remarkable reduction of time was achieved when immobilization was carried out by microwave irradiation. It took only 50 s to immobilize oxalate oxidase in the activated cuvet when exposed to microwaves in a domestic microwave oven. Immobilized-oxalate oxidase was stable, reusable and conveniently used for determination of urinary oxalate for diagnosis of primary hyperoxaluria and calcium oxalate nephrolithaiasis. Detection of human IgG involving ELISA in antihuman IgG-immobilized cuvet showed the feasibility of the cuvet in other immunoassays.

Oxalate decarboxylase (OxDC) catalyzes the conversion of oxalate into CO(2) and formate using a catalytic mechanism that remains poorly understood. The Bacillus subtilis enzyme is composed of two cupin domains, each of which contains Mn(II) coordinated by four conserved residues. We have measured heavy atom isotope effects for a series of Bacillus subtilis OxDC mutants in which Arg-92, Arg-270, Glu-162, and Glu-333 are conservatively substituted in an effort to define the functional roles of these residues. This strategy has the advantage that observed isotope effects report directly on OxDC molecules in which the active site manganese center(s) is (are) catalytically active. Our results support the proposal that the N-terminal Mn-binding site can mediate catalysis, and confirm the importance of Arg-92 in catalytic activity. On the other hand, substitution of Arg-270 and Glu-333 affects both Mn(II) incorporation and the ability of Mn to bind to the OxDC mutants, thereby precluding any definitive assessment of whether the metal center in the C-terminal domain can also mediate catalysis. New evidence for the importance of Glu-162 in controlling metal reactivity has been provided by the unexpected observation that the E162Q OxDC mutant exhibits a significantly increased oxalate oxidase and a concomitant reduction in decarboxylase activities relative to wild type OxDC. Hence the reaction specificity of a catalytically active Mn center in OxDC can be perturbed by relatively small changes in local protein environment, in agreement with a proposal based on prior computational studies.

The performance of enzymatic biosensors is highly dependent on the immobilization conditions of the enzyme. Immobilizing oxalate oxidase in a mucin/carbopol gel results in an amperometric electrode for oxalate sensing, with high stability and a much longer linear range than with albumin as support polymer. By way of a comparative analysis, we show in this paper how the physicochemical properties of the support may contribute to determine the analytical response of this type of sensor. Matrices based in mucin, carbopol, blends of mucin and carbopol, and albumin, all slightly crosslinked with glutaraldehyde, were analyzed. The swelling, rheological, diffusion and dielectric properties of the gels were studied as a function of pH. The results demonstrate that mucin induces high control for oxalate diffusion and that the interactions between mucin and carbopol molecules produce a notable gel strengthening that is reflected on the viscoelastic behavior and on the relatively smaller swelling index of the mucin/carbopol gels. The gel with a 70/30 ratio of mucin/carbopol concentrations presents the maximum rheological synergism and the minimum relative swelling index. Carbopol is responsible of the improved stability and strength of this matrix while mucin is accountable for its dielectric and diffusion behavior.

High-level secretory expression of wheat (Triticum aestivum) germin/oxalate oxidase was achieved in Pichia pastoris fermentation cultures as an alpha-mating factor signal peptide fusion, based on the native wheat cDNA coding sequence. The oxalate oxidase activity of the recombinant enzyme is substantially increased (7-fold) by treatment with sodium periodate, followed by ascorbate reduction. Using these methods, approximately 1 g (4x10(4) U) of purified, activated enzyme was obtained following eight days of induction of a high density Pichia fermentation culture, demonstrating suitability for large-scale production of oxalate oxidase for biotechnological applications. Characterization of the recombinant protein shows that it is glycosylated, with N-linked glycan attached at Asn47. For potential biomedical applications, a nonglycosylated (S49A) variant was also prepared which retains essentially full enzyme activity, but exhibits altered protein-protein interactions.

Oxalate oxidase (EC 1.2.3.4) catalyzes the oxidative cleavage of oxalate to carbon dioxide and hydrogen peroxide. In this study, unusual nonstoichiometric burst kinetics of the steady state reaction were observed and analyzed in detail, revealing that a reversible inactivation process occurs during turnover, associated with a slow isomerization of the substrate complex. We have investigated the underlying molecular mechanism of this kinetic behavior by preparing recombinant barley oxalate oxidase in three distinct oxidation states (Mn(II), Mn(III), and Mn(IV)) and producing a nonglycosylated variant for detailed biochemical and spectroscopic characterization. Surprisingly, the fully reduced Mn(II) form, which represents the majority of the as-isolated native enzyme, lacks oxalate oxidase activity, but the activity is restored by oxidation of the metal center to either Mn(III) or Mn(IV) forms. All three oxidation states appear to interconvert under turnover conditions, and the steady state activity of the enzyme is determined by a balance between activation and inactivation processes. In O2-saturated buffer, a turnover-based redox modification of the enzyme forms a novel superoxidized mononuclear Mn(IV) biological complex. An oxalate activation role for the catalytic metal ion is proposed based on these results.

Oxalate oxidase (EC 1.2.3.4) catalyzes the conversion of oxalate and dioxygen to hydrogen peroxide and carbon dioxide. In this study, glycolate was used as a structural analogue of oxalate to investigate substrate binding in the crystalline enzyme. The observed monodentate binding of glycolate to the active site manganese ion of oxalate oxidase is consistent with a mechanism involving C-C bond cleavage driven by superoxide anion attack on a monodentate coordinated substrate. In this mechanism, the metal serves two functions: to organize the substrates (oxalate and dioxygen) and to transiently reduce dioxygen. The observed structure further implies important roles for specific active site residues (two asparagines and one glutamine) in correctly orientating the substrates and reaction intermediates for catalysis. Combined spectroscopic, biochemical, and structural analyses of mutants confirms the importance of the asparagine residues in organizing a functional active site complex.

The synthesis and electrochemical characterization of a new organic/inorganic hybrid material was performed by combining polypyrrole and a hexacyano metalate (nickel hexacyanoferrate (NiHCNFe)) aiming to obtain an electrocatalyst for H2O2 reduction in the presence of either Na+ or K+ ions. The use of this material as a redox mediator in an oxalate biosensor based on the immobilization of oxalate oxidase enzyme was also discussed. The electrochemical properties of the hybrid material were investigated by using impedance measurements and compared with those of the nickel hexacyanoferrate film alone. The electrocatalytic properties of the hybrid for reducing H2O2, in the presence of both Na+ and K+ ions, are higher than those of the NiHCNFe film due to the presence of polypyrrole chains that enhances the electronic conductivity of the material.

NAD-dependent formate dehydrogenase (FDH, EC 1.2.1.2.) was purified 34-fold with 5.8% yield as an electrophoretically homogeneous protein from the white-rot fungus Ceriporiopsis subvermispora. The native enzyme has a molecular mass of 84 kDa consisting of two identical subunits as a homodimer. The K m values for formate and NAD were found to be 2.5 mM and 28 μM, respectively, at the optimum pH 6.5. The enzyme activity was inhibited by NADH, ADP, and ATP. The kinetic analysis indicated that the enzyme reaction proceeded by the ordered Bi Bi mechanism. Oxalate was found to be catabolized by mediations of oxalate decarboxylase (ODC, EC 4.1.1.2) and FDH during the vegetative growth of C. subvermispora, while it was oxidized by oxalate oxidase (OXO, EC 1.2.3.4) at the later stage of the cultivation. A possible role of the two-oxalate decomposing systems with ODC and OXO was discussed in relation to carbon metabolism of lignin-degrading basidiomycetes.

An enzymatic amperometric electrode with extended analytical range and improved stability for oxalate determination has been developed. Glutarlaldehyde/mucin/carbopol matrix was used for the crosslinking of the enzyme between polymeric membranes to form a classical laminate construction (sandwich) and compared with the glutaraldehyde/mucin/enzyme and glutaraldehyde/albumin/enzyme. The use of a sulphonated membrane as internal membrane allowed rejection of the most important electrooxidable urine interferents. The recovery assays were highly satisfactory. The wide linear response in the range 2–400 μM after 1/10 urine dilution (corresponding to 20–4000 μM) made it suitable for clinical range. High correlation with the standard spectrophotometric method was obtained ( r 2 = 0.98, y = 0.89 x , n = 25).

Oxalate degrading enzymes have a number of potential applications, including medical diagnosis and treatments for hyperoxaluria and other oxalate-related diseases, the production of transgenic plants for human consumption, and bioremediation of the environment. This review seeks to provide a brief overview of current knowledge regarding the major classes of enzymes and related proteins that are employed in plants, fungi, and bacteria to convert oxalate into CO 2 and/or formate. Not only do these enzymes employ intriguing chemical strategies for cleaving the chemically unreactive C C bond in oxalate, but they also offer the prospect of providing new insights into the molecular processes that underpin the evolution of biological catalysts.

Oxalate decarboxylase (EC 4.1.1.2) catalyzes the conversion of oxalate to formate and carbon dioxide and utilizes dioxygen as a cofactor. By contrast, the evolutionarily related oxalate oxidase (EC 1.2.3.4) converts oxalate and dioxygen to carbon dioxide and hydrogen peroxide. Divergent free radical catalytic mechanisms have been proposed for these enzymes that involve the requirement of an active site proton donor in the decarboxylase but not the oxidase reaction. The oxidase possesses only one domain and manganese binding site per subunit, while the decarboxylase has two domains and two manganese sites per subunit. A structure of the decarboxylase together with a limited mutagenesis study has recently been interpreted as evidence that the C-terminal domain manganese binding site (site 2) is the catalytic site and that Glu-333 is the crucial proton donor (Anand, R., Dorrestein, P. C., Kinsland, C., Begley, T. P., and Ealick, S. E. (2002) Biochemistry 41, 7659-7669). The N-terminal binding site (site 1) of this structure is solvent-exposed (open) and lacks a suitable proton donor for the decarboxylase reaction. We report a new structure of the decarboxylase that shows a loop containing a 3(10) helix near site 1 in an alternative conformation. This loop adopts a "closed" conformation forming a lid covering the entrance to site 1. This conformational change brings Glu-162 close to the manganese ion, making it a new candidate for the crucial proton donor. Site-directed mutagenesis of equivalent residues in each domain provides evidence that Glu-162 performs this vital role and that the N-terminal domain is either the sole or the dominant catalytically active domain.

Oxalate oxidase catalyses the degradation of oxalic acid to carbon dioxide and hydrogen peroxide and is of commercial importance for clinical analyses of oxalate in biological samples. Novel potential applications for oxalate oxidase include the prevention of the formation of calcium oxalate incrusts in pulp and paper manufacture and rapid determination of oxalic acid in process waters. The potential in using oxalate-degrading enzymes in industrial processes increases the interest in finding systems for heterologous expression. Oxalate oxidase from barley is a secreted multimeric glycosylated manganese-containing enzyme with several disulfide bridges, which have been found to be essential for the catalytic activity. Attempts to achieve expression of active heterologous oxalate oxidase in bacteria have up to now met little success. In this study, one oxalate-oxidase-encoding cDNA from barley and two from wheat were cloned and tested with regard to expression in Escherichia coli. The results suggest that the selection of a novel commercially available E. coli host strain, which has the ability to form disulfide bridges in heterologous proteins expressed in its cytoplasm, was important for successful expression. Although a considerable part of the heterologous protein was produced in an insoluble and inactive form, this strain, E. coli Origami B(DE3), in addition yielded soluble and active barley and wheat oxalate oxidase. One of the wheat cDNAs, Ta(M)OXO1, gave three-fold higher activity than the barley cDNA, Hv(H)OXO1, while the other wheat cDNA, Ta(M)OXO2, gave no detectable activity. This indicates that the choice of cDNA was also critical despite the high identity between the cDNAs and the encoded polypeptides (88-89% on the nucleotide level and 88-92% on the amino-acid level). Gel filtration of cell extracts containing heterologous barley and wheat oxalate oxidase resulted in an increase in the activity. This indicates that low molecular weight inhibitory compounds were present in the E. coli lysates but could be removed by the introduction of a purification step.

A new method for rapid determination of oxalic acid was developed using oxalate oxidase and a biosensor based on SIRE (sensors based on injection of the recognition element) technology. The method was selective, simple, fast, and cheap compared with other present detection systems for oxalate. The total analysis time for each assay was 2-9 min. A linear range was observed between 0 and 5 mM when the reaction conditions were 30 degrees C and 60 s. The linear range and upper limit for concentration determination could be increased to 25 mM by shortening the reaction time. The lower limit of detection in standard solutions, 20 microM, could be achieved by means of modification of the reaction conditions, namely increasing the temperature and the reaction time. The biosensor method was compared with a conventional commercially available colorimetric method with respect to the determination of oxalic acid in urine samples. The urine oxalic acid concentrations determined with the biosensor method correlated well (R=0.952) with the colorimetric method.

Background: Oxalate generation at pH-values above 5.0 and an oxalate–protein binding in acidified plasma would appear to complicate the determination of oxalate in plasma. Methods: To avoid complex sample preparation we used a high-performance liquid chromatographic system with an inline enzyme reactor (HPLC-ER) containing immobilised oxalate oxidase. The detection limit was 0.68 μmol/l. Blood was drawn in lithium–heparin vessels and immediately centrifuged at 4 °C. The yielded plasma was ultrafiltered using a Centrisart-I-tube. To inhibit oxalate generation by ascorbic acid, the ultrafiltrate was acidified with 1 mol/l hydrochloric acid during ultrafiltration at 4 °C. The liquid thus yielded was used for HPLC-ER analysis. Blood samples were obtained from 133 healthy adults (63 men, 70 women, aged 20–94 years) with no history of renal disorder and from 79 patients (53 men, 26 women, aged 19–77 years) with a history of calcium oxalate stone formation. Results: Mean plasma oxalate was 2.65±2.31 μmol/l for healthy subjects and 4.21±0.56 μmol/l for stone formers. Conclusions: Analysis yielded no significant differences between males and females. A correlation between age and plasma oxalate was found for the healthy adults (p

The strategy of targeting expression of a constitutively regulated gene to generate H 2 O 2 in the extracellular matrix, to reduce herbivory of the European corn borer (ECB) [ Ostrinia nubilalis (Hubner)] was tested by transforming corn with germin, a wheat oxalate oxidase (OXO) gene, regulated by the rice actin promoter elements (pAct-OXO). With two independent transformation events, enzyme activity was stable over seven generations of backcrossing into three maize inbred lines. Enzyme activity remained associated with the cell wall debris fraction of water extracted tissues. Leaf tissue of the germin transgenics had elevated levels of H 2 O 2 . In vitro leaf feeding bioassays demonstrated that ECB larvae feeding was significantly reduced and larval growth and development were delayed on all ECB infested germin transgenic lines. This reduced ECB feeding was confirmed under field conditions. Most significantly, stalk tunneling damage, measured at plant harvest, was substantially reduced in all germin transgenic lines. The reduction of tunneling by 50% in the transgenic lines is indicative of lower levels of ECB survival which should be significant in ECB epidemiology. Possible mechanisms of resistance include modifications in plant cell wall chemistry, activation of pathogen resistance genes and effects of H 2 O 2 and germin on insect physiology are discussed.

This work describes a simple and low cost methodology for oxalate determination in food samples, which employs a bromothymol blue-based pH optode for the determination of CO2 generated in the enzymatic reaction between oxalic acid and oxalate oxidase. The enzyme was immobilised on barley seeds, together with catalase enzyme, and placed in a stirring bar type enzymatic reactor. The system showed a linear response range from 0.0080 up to 0.100 mol l −1 , when the measurements were carried out in 0.050 mol l −1 succinate buffer at pH 4.0 and 25 ◦ C. In these conditions, the lifetime of the system was about 120 h, with a relative standard deviation

The Bacillus subtilis oxalate decarboxylase (EC ), YvrK, converts oxalate to formate and CO(2). YvrK and the related hypothetical proteins YoaN and YxaG from B. subtilis have been successfully overexpressed in Escherichia coli. Recombinant YvrK and YoaN were found to be soluble enzymes with oxalate decarboxylase activity only when expressed in the presence of manganese salts. No enzyme activity has yet been detected for YxaG, which was expressed as a soluble protein without the requirement for manganese salts. YvrK and YoaN were found to catalyze minor side reactions: oxalate oxidation to produce H(2)O(2); and oxalate-dependent, H(2)O(2)-independent dye oxidations. The oxalate decarboxylase activity of purified YvrK was O(2)-dependent. YvrK was found to contain between 0.86 and 1.14 atoms of manganese/subunit. EPR spectroscopy showed that the metal ion was predominantly but not exclusively in the Mn(II) oxidation state. The hyperfine coupling constant (A = 9.5 millitesla) of the main g = 2 signal was consistent with oxygen and nitrogen ligands with hexacoordinate geometry. The structure of YvrK was modeled on the basis of homology with oxalate oxidase, canavalin, and phaseolin, and its hexameric oligomerization was predicted by analogy with proglycinin and homogentisate 1,2-dioxygenase. Although YvrK possesses two potential active sites, only one could be fully occupied by manganese. The possibility that the C-terminal domain active site has no manganese bound and is buried in an intersubunit interface within the hexameric enzyme is discussed. A mechanism for oxalate decarboxylation is proposed, in which both Mn(II) and O(2) are cofactors that act together as a two-electron sink during catalysis.

Germins and germin-like proteins (GLPs) constitute a large and highly diverse family of ubiquitous plant proteins. The name germin was given because the first described member of the family, wheat germin, was discovered in germinating wheat grains. However, it is now known that proteins from this family exist in all organs and developmental stages and that several are also involved in the response to various stress conditions. It is thus increasingly obvious that germins and GLPs participate in many processes that are important for plant development and defence. However, their exact participation in these processes generally remains obscure so the main challenge now is to determine the precise activities and functions of all germins and GLPs. Molecular and biochemical studies are contributing to a better understanding of this protein family and much data has accumulated in the last 2 years. All species possess a wide range of GLPs and each GLP gene is subjected to a tight regulation. All germins and GLPs are glycoproteins somehow associated with the extracellular matrix. More specifically, three classes of functions are starting to be recognized for these proteins: some possess an enzymatic activity (oxalate oxidase or superoxide dismutase), others seem to be structural proteins while some others act as receptors. To explain the diversity and ubiquitousness of germins and GLPs, we propose that most, if not all, of them carry out a combination of different functions.

The present project aimed to isolate testa-, pericarp- and epicarp-specific gene promoters for the developing caryopsis of barley (Hordeum vulgare L.). These might be applied in transgenic plants to express antifungal agents or modify metabolic pathways. A testa-specific 379-nucleotide fragment was cloned by differential amplification and used to screen a bacterial artificial chromosome (BAC) library of 6.3 haploid genome equivalents. Fifty-three clones containing genes encoding for proteins of the germin family were found. Characterization of the clones identified a minimum of six seed coat- and eight leaf-specific germin genes. Four seed coat- and one leaf-specific genes were sequenced. The deduced primary structure of the proteins revealed a remarkable conservation of the manganese(II) binding His and Glu residues and β-barrel secondary structure of oxalate oxidase – also in barley, wheat, rice and Arabidopsis germins, for which an enzymatic activity has not yet been identified. The oxalate oxidase and germins of barley and other species are synthesized with a conserved pre-sequence of 23 or 24 amino acids for targeting into the cell wall. β-Glucuronidase expression with the barley germin F gene promoter occurs specifically in the testa and epicarp of the developing barley caryopsis, while expression with the B gene promoter is restricted to the testa. Oxalate oxidase activity is prominent in the epicarp and the root tips of the developing embryo. A family tree based on primary structure homologies of germins distinguishes three groups: oxalate oxidases, leaf-specific germins and seed coat-specific germins.

Oxalate oxidase activity was detected in the cell wall fraction isolated from maize roots (Zea mays L.). The enzyme was active at acidic pH with optimal activity at pH 3.2. It was thermally extremely stable and resistant to high salt concentration, SDS and pepsin. The enzyme activity was inhibited by sulphydryl reagents 2-mercaptoethanol (2-ME), N-ethyl maleimide (NEM) and dithiotreitol (DTT), but was insensitive to EDTA, KCN and metal ions. Measurements of enzyme activity were performed using colorimetric assay of H2O2, as well as polarographic detection of O2 consumption. Maximal activity was obtained with 5 mM oxalic acid for the colorimetric method, and 10 mM oxalic acid for the polarographic method. Both methods were applicable in oxalate oxidase characterization, the polarographic method being more suitable under conditions of H2O2 interaction with some of the analyzed substances. © 2000 Elsevier Science Ireland Ltd. All rights reserved.

We have reported a simple method of determination of plasma oxalate using a Cl(-) and NO(3)(-) insensitive oxalate oxidase purified from grain sorghum leaf and commercially available peroxidase from horseradish [Pundir et al., Ind. J. Biochem. Biophys., 35 (1998) 120-122]. The present report describes the immobilization of both the enzymes onto alkylamine glass, their kinetic properties and application for discrete analysis of plasma oxalate. In the analytic method, H(2)O(2) generated from plasma oxalate by immobilized oxalate oxidase is measured colorimetrically at 520 nm by oxidative coupling with 4-aminophenazone, and phenol catalyzed by immobilized peroxidase. The minimum detection limit of the method is 2.5 micromol/l. Analytic recovery of added oxalate in plasma was 89. 5+/-4.1% (mean+/-S.D.). The within and between day CV for plasma oxalate measurement were9.37 and11.0%, respectively. The normal range of plasma oxalate as measured by the present method was 3.6 to 5.7 micromol/l. The method is not only free from interference by plasma Cl(-) and NO(3)(-) but also provides the reuse of glass beads and thus reduces the cost of analysis for routine.

A study of selected transcripts in rice leaves was performed using a number of cDNA clones from barley as probes. The transcripts for the pathogenesis–related proteins, PR-1, PR-2, PR-3, PR-4 and PR-5, and for a peroxidase were found to accumulate strongly in response to the non-rice pathogen Bipolaris sorokiniana and moderately in response to a virulent isolate of Pyricularia oryzae (Magnaporthe grisea) and u.v. light. A weak accumulation was observed in response to an avirulent isolate of P. oryzae. PR-protein transcript accumulation was seen 12 h after inoculation with B. sorokiniana and after exposure to u.v. light. These transcripts reached maximum accumulation levels at 24 h and all declined thereafter with the exception of the PR-4 transcript in response to B. sorokiniana. Maximum accumulation of the peroxidase transcript occurred at 12 h in response to both B. sorokiniana and u.v. light. These PR-protein and peroxidase transcripts were later expressed markedly in response to avirulent and virulent isolates of P. oryzae. They were barely detectable at the early time points, while maximum transcript levels occurred towards the end of the experiment, i.e. 120 h after inoculation. Interestingly, all these transcripts accumulated to a higher level in the compatible than in the incompatible interactions between rice and P. oryzae. Transcripts for chalcone synthase, oxalate oxidase and an oxalate oxidase-like protein were not detectable using the heterologous barley clones, whilst 14-3-3 protein and O -methyltransferase transcripts were seen to be constitutively expressed.

A novel extracellular Mn-superoxide dismutase (SOD) was isolated from a moss, Barbula unguiculata. The SOD was a glycoprotein; the apparent molecular mass of its native form was 120 kDa, as estimated by gel filtration chromatography, and that of its monomer was 22,072 Da, as estimated by time of flight mass spectroscopy. The protein had manganese with a stoichiometry of 0.80 Mn/monomer. The cDNA clone for a gene encoding the extracellular Mn-SOD was isolated. Sequence analysis showed that it has a strong similarity to germin (oxalate oxidase) and germin-like proteins (GLPs) of several plant species and possesses all the characteristic features of members of the germin family. The clone encoding this extracellular Mn-SOD was therefore designated B. unguiculata GLP (BuGLP). BuGLP had no oxalate oxidase activity. In addition, the cDNA for a gene encoding the moss mitochondrial Mn-SOD was isolated. Its amino acid sequence had little similarity to that of BuGLP, even though a close similarity was observed among the mitochondrial Mn-SODs of various organisms. BuGLP was the first germin-like protein that was really demonstrated to be a metalloprotein with Mn-SOD activity but no oxalate oxidase activity.

Summary Silene vulgaris ssp. humilis , a heavy metal tolerant plant growing on the polluted soil of a medieval copper mining dump, accumulates considerable amounts of heavy metals (HM) in its roots and shoots. The intracellular distribution of HMs in the leaves was investigated by conventional and analytical (EDX, ESI, EELS) electron microscopy. Part of the HMs, Fe, Cu, and Zn occur as crystalline compounds on the surface of the leaves. The epidermal cell walls accumulate Fe, Ni, Cu, AI, Sn, and Zn. Cu within the cell walls is tightly bound to a protein with oxalate oxidase activity, evidencing a high homology to germin. Zn and Sn are accumulated in the cell walls as silicate. Cytoplasm and organelles contain only traces of Cu and Sn, while in the vacuoles no HMs are detected. In the epidermal cell walls, intercellular spaces, and in vacuoles there are high concentrations of Si, forming crystal-like structures. EELS and quantum-chemical calculations reveal these structures as SiO 2 . The role of Si in the HM-tolerance of Silene is discussed.

A membrane bound oxalate oxidase has been purified to apparent homogeneity from mature leaves of the wild herb Amaranthus spinosus . The M r of the enzyme was ca. 130 kDa by Sephadex G-200 gel filtration and 65 kDa by SDS disc electrophoresis indicating two subunits of identical M r . The enzyme showed optimum activity at pH 3.5 when incubated at 40°C for 5 min. The energy of activation of the enzyme was 15.25 kcal mol −1 . The K m for oxalate and V max of the enzyme reaction were 2.16×10 −3 M and 0.18 μmol min −1 ml −1 , respectively. Sodium diethyl dithiocarbamate and sodium azide inhibited the enzyme while cysteine caused slight inhibition. Metals and flavins had no effect on the enzyme.

Oxalate oxidase (OXO) was chemically modified using amino acid-specific reagents. The modification reactions were monitored spectrophotometrically, to follow the progress of labeling, and catalytically, to assess the effect of labeling on the enzyme function. The enzyme does not bear arginines essential for activity, since 2,3-butanedione and cyclohexanodione, although they modify the enzyme (after chromatographic analysis), have no effect on its activity. Incubation of urea-pretreated OXO withN-acetylimidazole leads to labeling all 10 tyrosines without affecting the enzyme activity, thus suggesting that OXO does not have tyrosines essential for activity. However, OXO modification with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide followed by kinetic analysis, leads to the conclusion that the enzyme possesses one carboxylate essential for activity. When using the modifier 2,4,6-trinitrobenzene sulfonic acid (TNBS), while 28 of the total 45 lysines are labeled within 3 h (the first 5 reacting lysines of the homopentametic enzyme are modified at a faster rate than the others), the enzyme rapidly loses 90% of its activity in the first 2 min, a period during which only one lysine is being labeled. Complete enzyme inactivation with TNBS is observed after approximately 8 min, when 5 lysines are being labeled. The modification of the first lysine also triggers the dissociation of native OXO to its subunits (after SDS–PAGE analysis), a phenomenon not observed with the other modifiers. These findings indicate that OXO bears a lysine per monomer, essential for enzyme activity. When using 5,5-dithio-bis-(2-nitrobenzoic)acid to determine the number of disulfide bonds, in the presence of NaBH4, 10 sulfhydryls are determined, but in the absence of reducing agent, none are determined. Further, chloro-mercuribenzoate does not inactivate OXO but β-mercaptoethanol does. Therefore, the sulfhydryls in OXO are not free but form disulfide bonds essential for activity. Furthermore, the metallo-chelating agents HgCl2and 8-hydroxychinolin inactivate the enzyme, suggesting that barley root oxalate oxidase is a metalloenzyme. It is possible that the metal(s) are involved in the oxidative mechanism since the enzyme does not bear prosthetic groups such as FAD and FMN.

A number of barley cDNAs isolated by differential screening represent gene transcripts which accumulate during fungal pathogen attack. The encoded proteins comprise species commonly encountered in plant–pathogen interactions, e.g. PR proteins, but also protein species not previously reported from plant-pathogen interactions: a novel flavonoidO-methyltransferase, a 14-3-3 protein, an endoplasmatic chaperone (GRP94), an oxalate oxidase, an oxalate oxidase-like protein, and some species with no known homologues. One of these latter sequences BH72-Q3, encoding a small proline-rich protein, is presented here. Transcripts of 15 gene families accumulated in the leaves following inoculation withBlumeria graminisf.sp.hordei(syn.Erysiphe graminisf.sp.hordei) in both compatible and incompatible interactions. During the first 24 h after inoculation no clear differences were observed among different interaction types. At later stages transcript accumulation reflected the different infection types, with strong late accumulation in compatible interactions, a somewhat earlier accumulation in an incompatible multi-cell HR interaction, and only weak accumulation in an incompatible single cell HR interaction. Experiments using isolated epidermal strips and the residual mesophyll material showed that the oxalate oxidase-like gene transcript accumulated exclusively in the epidermis, whereas the 14-3-3, GRP94, and BH6-12 transcripts accumulated in both epidermis and mesophyll. All other gene transcripts appeared to accumulate preferentially in the mesophyll cells, however, with increased epidermal accumulation late after inoculation. The exception is the oxalate oxidase transcript, which accumulated exclusively in the mesophyll. The response of the barley plant to attack by the mildew fungus thus appears to consist of a complex of interconnected reactions in the epidermis and the underlying mesophyll cells.

An enzyme electrode has been developed for the simplified determination of oxalate in human urine. Oxalate oxidase has been co-immobilised with bovine serum albumin (BSA) using glutaraldehyde between polycarbonate or haemodialysis external membranes and an internal cellulose acetate membrane to form a classical oxidase enzyme laminate construction. The underlying cellulose acetate permselective barrier confers the required selectivity for H 2 O 2 over the interferent constituents of urine. Prior dilution and acidification of urine samples has enabled optimisation of enzyme activity and therefore electrode response characteristics for low level oxalate assay. Sequestering of singly and doubly charged cations in urine by ethylenediaminetetraacetic acid (EDTA) has ensured release of bound oxalate, so permitting near total (> 98%) determination of urinary oxalate. An outer substrate diffusion limiting membrane has proved critical to an electrode linear response within the clinical concentration range. Determination of human urinary oxalate in the concentration range 2–200 μM in diluted urine (corresponding to 80–800 μM oxalate in undiluted urine) has been possible. Results correlate well ( r 2 = 0.971; y = 1.11 x + 4.20; n = 20) with standard spectrophotometric determinations ( x ) performed within a hospital laboratory.

Novel uric acid and oxalic acid sensors have been developed by immobilizing uricase and oxalate oxidase to form bio-thermochips. Uric acid is determined in the range 1–6 mM and oxalic acid in the range 0.2–0.8 mM. To measure the kidney calculus indices (uric acid and oxalic acid) simultaneously, the feasibility of integrating two two sensors has been examined. The pH dependence of the oxalic acid sensor, the effect of uric acid on the oxalic acid sensor and the effect of oxalic acid on the uric acid sensor are examined. Although the optimum pH for oxalate oxidase is 5, the response of the oxalic acid sensor in pH 8 buffer is high enough for the determination of oxalic acid. Uric acid and oxalic acid do not interfere with the determination of each other's concentration using the oxalic acid and uric acid sensors. Therefore, construction of a multi-functional bio-thermochip system for the determination of kidney calculus indices, uric acid and oxalic acid, should be made possible by integrating the two bio-thermochips.