The computer model supports the following reaction mechanism: The Oxo-Mn(2)-oxalate complex exists as a mixture of five-and six-coordinate species. The form with coordinatively unsaturated Mn(2) site reacts with dioxygen on the quartet potential energy surface. In this step, the proton from oxalate monoanion is transferred to dioxygen through the first-shell glutamate. The proton-transfer triggers the C-C bond cleavage, and the electron follows the proton. Simultaneously, the second electron, necessary to produce the peroxo species, is provided by manganese. This step, which is also rate-limiting, yields the first CO2 molecule and the reactive intermediate in which the formyl radical anion coordinates the high-spin Mn(3). The quartet to sextet spin transition, which involves a small apparent barrier, allows for the formyl radical -> Mn(3) electron transfer. This step leads to the product-active site complex, which upon protonation decays to H2O2, CO2, and the active site is then ready to begin the next catalytic cycle
treatment of the periodate-oxidized enzyme with ascorbate results in a substantioal decrease in absorption, forming a complex that is spectroscopically identified as a Mn3+ species. Mn3+ form has a 5fold higher specific activity than native recombinant oxalate oxidase.
titration of oxalate oxidase with sodium periodate results in nearly stoichometric oxidation of the enzyme to an intensely colored yellow complex, whose complete spectroscopic characterization lead to assignment to a superoxidized Mn5+ complex. Treatment of Mn2+ S49A oxalate oxidase generates the same yellow species as the glycosylated wild type enzyme. Mass spectra of isolated and periodate-treated oxalate oxidase are virtually identical, demonstating that no protein oxidation occurred. Peroxidate oxidation increases the specific activity about 5fold.
titration of periodate-oxidized oxalate oxidase with hydroxylamine completely eliminates the visible absorption, forming a homogeneous Mn2+ form of the enzyme. The fully reduced Mn2+ form lacks any detectable oxidase activity, reoxidation substantially restores the maximum activity.
the specific activity of oxalate oxidase is increased in seedlings grown in a NaCl containing medium compared to normal, which reveals the increased de novo synthesis of the enzyme to sustain oxalate egradation
oxalate oxidase activity is measured by oxygen uptake assay with a Clark oxygen electrode in a thermostated cell (25Â°C). The Km evalutated from initial velocity data exhibits a strong pH dependence with limiting slopes (versus pH) of 0.9 (below pH 4) and 1.5 (above pH 4)
asparagine mutant (N85A), thermostated (25Â°C) Clark oxygen electrode calibrated with the protocatechuic acid/protocatechuate dioxygenase reaction, pH 4, specific activity is higher in the presence of 1 M NH4Cl or 1 M NaCl and slightly lower when 1 M formamide is added
asparagine mutant (N75A),thermostated (25Â°C) Clark oxygen electrode calibrated with the protocatechuic acid/protocatechuate dioxygenase reaction, pH 4, specific activity is slightly higher in the presence of 1 M NH4Cl or 1 M NaCl and does not change when 1 M formamide is added
the effect of the isotopic composition of the solvent is investigated by assaying oxalate oxidase in buffer prepared form H2O/D20 mixtures.The limiting values of Vs (steady state rate) lead to an estimate of the overall solvent kinetic isotope effect kH2O/kD2O = 8.5 (k=burst rate constant)
in situ, max. rate of oxalate degradation in the liquid phase of a spinach (commercial frozen) preparation, the max. rate of oxalate degradation in spinach suspension and the solid phase is reached at pH 3.5
in situ, max. rate of oxalate degradation in the liquid phase of a spinach (commercial frozen) preparation, the rate of oxalate degradation in spinach suspension and the solid phase is lower. In fresh spinach (without any heat treatment) little oxalate degradation occurs
in situ, max. oxalate degradation in the liquid phase of a spinach (commercial frozen) preparation, the rate of oxalate degradation in spinach suspension and the solid phase is lower. In fresh spinach (without any heat treatment) little oxalate degradation occurs
cultivars PI 255956, PI 535278 (Tars-046A) and cv. Wolven Pole, oxalate concentration in infected (infected with Sclerotinia sclerotiorum) stems of Wolven Pole is higher than in PI 255956 and PI535278. Inoculated stems of Wolven Pole have oxalate oxidase, the Sclerotinia sclerotorum resistent lines PI 255959 and PI 535278 not. Phaseolus coccineus is not as oxalat sensitiv as Phaseolus vulgaris. Infection of Phaseolus coccineus with Sclerotinia scerotorum increased the levels of oxalate, with the highest concentration in Woven Pole
cvs. Huron (navy), Othello (pinto) and Newport (navy), Phaseolus vulgaris is more oxalate sensitive than Phaseolus coccineus, with Othello being the most sensitive, Huron the most tolerant, and Newport intermediate. Huron is more resistant to Sclerotinia sclerotiorum than the other two cultivars
transgenic potato plantlets expressing the barley oxalate oxidase enzyme, show a relatively higher salinity tolerance than the non-transgenic genotypes in vitro, but in the glasshouse the results are less consistent
When embryos are cultured together with endosperms (endosperm-supported culture, ES), the percentage of callus induction is significantly lower than that when embryos are cultured in the absence of endosperm (non-endosperm-supported culture, NES). The activity of oxalate oxsidase in the callus of ES culture is higher than that in the callus of NES culture, suggesting that the activity of oxalate oxidase may be a parameter for selection of calli with potential for plantlet regeneration
fresh or heat dried (55Â°C for 16 h, with T increased in 5Â°C steps to reach 75Â°C) radicles are used. Heat drying reduces the rate of oxalate degradation activity by approximately half. Deep-freezing the radicles to -20Â°C does not lead to any loss of oxalate degradation activity within 4 weeks of storage
the enzyme is capable of stimulating the ester-linked diferulic acid formation. The enzyme 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
the enzyme is not a disease resistance factor in rice. since transgenic rice plants with substantially higher enzyme activity are not more resistant to rice blast and bacterial blight than the wild type
hanging-drop vapor diffusion method at 18 Â°C using 1 micro l of protein (10-15 mg/ml) plus 1 micro l reservoir drops an 1-ml reservoirs, native crystals grown from 2.3M (NH4)2SO4 and 5% 2-propanol are rhombohedral, crystals of recombinant protein, grown from 10% polyethylene glycol 4000 and 0.1 M NaAc, pH 4.6, are tetragonal, Asn75-> Ala OXO are grown from 20% 2-propanol, 0.1 M NaAc, pH 4.6, 0.2 M CaCl2, can be cryo-cooled directly and are rhombohedral
metal oxide nanoparticles(NP)-bound enzyme retains more activity when subjected to thermal treatment at 70Â°C for 30 min., retention of activity in the increasing order being 54%, 65%, 76%, and 87% for native, ZnO NPs-, CuO NPs-, and MnO2 NPs-bound enzyme, respectively
into the pBI121 binary vector, the new construct pBI-OxO is transformed into Escherichia coli and then to Agrobacterium tumefaciens LBA4404, subsequently leaf discs of Nicotiana tabacum plants are transformed by culturing with Agrobacterium tumefaciens
transgenic potato plants (original potato plant = Solanum tuberosum L. cultivar Maris Brad and Desiree) expressing the oxalate oxidase enzyme are produced unsing Agrobacterium (Agrobacterium tumefaciens strain LBA4404)-mediated transformation