Mechanism of versatile peroxidase inactivation by Ca(2+) depletion

Versatile peroxidase (VP) from Bjerkandera adusta, as other class II peroxidases, is inactivated by Ca(2+) depletion. In this work, the spectroscopic characterizations of Ca(2+)-depleted VP at pH 4.5 (optimum for activity) and pH 7.5 are presented. Previous works on other ligninolytic peroxidases, such as lignin peroxidase and manganese peroxidase, have been performed at pH 7.5; nevertheless, at this pH these enzymes are inactive independently of their Ca(2+) content. At pH 7.5, UV-Vis spectra indicate a heme-Fe(3+) transition from 5-coordinated high-spin configuration in native peroxidase to 6-coordinated low-spin state in the inactive Ca(2+)-depleted form. This Fe(3+) hexa-coordination has been proposed as the origin of inactivation. However, our results at pH 4.5 show that Ca(2+)-depleted enzyme has a high spin Fe(3+). EPR measurements on VP confirm the differences in the Fe(3+) spin states at pH 4.5 and at 7.5 for both, native and Ca(2+)-depleted enzymes. In addition, EPR spectra recorded after the addition of H(2)O(2) to Ca(2+)-depleted VP show the formation of compound I with the radical species delocalized on the porphyrin ring. The lack of radical delocalization on an amino acid residue exposed to solvent, W170, as determined in native enzyme at pH 4.5, explains the inability of Ca(2+)-depleted VP to oxidize veratryl alcohol. These observations, in addition to a notorious redox potential decrease, suggest that Ca(2+)-depleted versatile peroxidase is able to form the active intermediate compound I but its long range electron transfer has been disrupted.     

Effect of strophanthin G on peroxidase oxidation kinetics of slowly oxidizable peroxidase substrates

Steady-state kinetics of thioproperazine, triftazine, aminazine, and o-dianisidine oxidation with hydrogen peroxide catalyzed by horseradish peroxidase were studied in the presence of strophanthin G. Values of the inhibition and activation constants (Ki, Ka) were determined in the pH range 5.0-7.5. At acidic pH, strophanthin G activated peroxidase during the oxidation of thioproperazine by the uncompetitive mechanism, and when triftazine was oxidized, the inhibition was noncompetitive. At pH > 6.0, the patterns of activation and inhibition changed to mixed-type during the peroxidase oxidation of thioproperazine and triftazine and to competitive inhibition of peroxidase with strophanthin G during the oxidation of aminazine. These effects are suggested to be due to an ionizable enzyme group of pK approximately 6.0. Strophanthin G inhibited free-radical oxidation of o-dianisidine via binding to the enzyme-substrate complex, preventing the generation of a stable semi-oxidized product of o-dianisidine, and thus inhibiting the enzyme by the anticompetitive mechanism. Mechanisms of oxidation of slowly and rapidly oxidizable substrates of peroxidase in the presence of strophanthin G are suggested.     

Manganese oxidation site in Pleurotus eryngii versatile peroxidase: a site-directed mutagenesis, kinetic, and crystallographic study

The molecular architecture of versatile peroxidase (VP) includes an exposed tryptophan responsible for aromatic substrate oxidation and a putative Mn2+ oxidation site. The crystal structures (solved up to 1.3 A) of wild-type and recombinant Pleurotus eryngii VP, before and after exposure to Mn2+, showed a variable orientation of the Glu36 and Glu40 side chains that, together with Asp175, contribute to Mn2+ coordination. To evaluate the involvement of these residues, site-directed mutagenesis was performed. The E36A, E40A, and D175A mutations caused a 60-85-fold decrease in Mn2+ affinity and a decrease in the Mn2+ oxidation activity. Transient-state kinetic constants showed that reduction of both compounds I and II was affected (80-325-fold lower k2app and 103-104-fold lower k3app, respectively). The single mutants retained partial Mn2+ oxidation activity, and a triple mutation (E36A/E40A/D175A) was required to completely suppress the activity (<1% kcat). The affinity for Mn2+ also decreased ( approximately 25-fold) with the shorter carboxylate side chain in the E36D and E40D variants, which nevertheless retained 30-50% of the maximal activity, whereas similar mutations caused a 50-100-fold decrease in kcat in the case of the Phanerochaete chrysosporium manganese peroxidase (MnP). Additional mutations showed that introduction of a basic residue near Asp175 did not improve Mn2+ oxidation as found for MnP and ruled out an involvement of the C-terminal tail of the protein in low-efficiency oxidation of Mn2+. The structural and kinetic data obtained highlighted significant differences in the Mn2+ oxidation site of the new versatile enzyme compared to P. chrysosporium MnP.     

Fluorophenol oxidation by a fungal chloroperoxidase

Caldariomyces fumago chloroperoxidase degrades monofluorophenols at both pH 3 and pH 6. 4-Fluorophenol is most readily degraded and its oxidation is most efficient at pH 6. GC-MS analyses of the reaction products revealed compounds relating to the reaction of fluorophenol radical. The degradation of fluorinated compounds is of significant environmental interest and this versatile enzyme may by employed to treat contaminated soil or water prior to discharge.     

Optimization of extracellular fungal peroxidase production by 2 Coprinus species

Optimum culture conditions for the batch production of extracellular peroxidase by Coprinus cinereus UAMH 4103 and Coprinus sp. UAMH 10067 were explored using 2 statistical experimental designs, including 2-level, 7-factor fractional factorial design and 2-factor central composite design. Of the 7 factors examined in the screening study, the concentrations of carbon (glucose) and nitrogen (peptone or casitone) sources showed significant effects on the peroxidase production by Coprinus sp. UAMH 10067. The optimum glucose and peptone concentrations were determined as 2.7% and 0.8% for Coprinus sp. UAMH 10067, and 2.9% and 1.4% for C. cinereus UAMH 4103, respectively. Under the optimized culture condition the maximum peroxidase activity achieved in this study was 34.5 U x mL(-1) for Coprinus sp. UAMH 10067 and 68.0 U x mL(-1) for C. cinereus UAMH 4103, more than 2-fold higher than the results of previous studies.     

Directed evolution of a fungal peroxidase

The Coprinus cinereus (CiP) heme peroxidase was subjected to multiple rounds of directed evolution in an effort to produce a mutant suitable for use as a dye-transfer inhibitor in laundry detergent. The wild-type peroxidase is rapidly inactivated under laundry conditions due to the high pH (10.5), high temperature (50 degrees C), and high peroxide concentration (5-10 mM). Peroxidase mutants were initially generated using two parallel approaches: site-directed mutagenesis based on structure-function considerations, and error-prone PCR to create random mutations. Mutants were expressed in Saccharomyces cerevisiae and screened for improved stability by measuring residual activity after incubation under conditions mimicking those in a washing machine. Manually combining mutations from the site-directed and random approaches led to a mutant with 110 times the thermal stability and 2.8 times the oxidative stability of wild-type CiP. In the final two rounds, mutants were randomly recombined by using the efficient yeast homologous recombination system to shuffle point mutations among a large number of parents. This in vivo shuffling led to the most dramatic improvements in oxidative stability, yielding a mutant with 174 times the thermal stability and 100 times the oxidative stability of wild-type CiP.     

白腐菌产锰过氧化物酶培养基的优化

黄孢原毛平革菌(Phanerochaete Chrysosporium)5．776在初始发酵培养基中产胞外锰过氧化物酶活力极低。为了显著提高锰过氧化物酶活力,对初始发酵培养基进行优化。通过调整培养基中碳源、氮源种类和含量,吐温80添加量,Mn^2 终浓度,静置培养温度、时间,采用分光光度计法测定酶活力,发现黄孢原毛平革菌在限氮高锰培养基中产生较高的锰过氧化物酶。静置液体培养的优化条件是：葡萄糖10g／L;酒石酸铵2mmol／L;吐温80 lg／L;Mn^2 9．9μg／L;于34℃静置培养5d;产MnP活力达1200U／L,比优化前提高了近17倍。     

Oxidation of pharmaceutically active compounds by a ligninolytic fungal peroxidase

Pharmaceuticals are an important group of emerging pollutants with increasing interest due to their rising consumption and the evidence for ecotoxicological effects associated to trace amounts in aquatic environments. In this paper, we assessed the potential degradation of a series of pharmaceuticals: antibiotics (sulfamethoxazole), antidepressives (citalopram hydrobromide and fluoxetine hydrochloride), antiepileptics (carbamazepine), anti-inflammatory drugs (diclofenac and naproxen) and estrogen hormones (estrone, 17β-estradiol, 17α-ethinylestradiol) by means of a versatile peroxidase (VP) from the ligninolytic fungus Bjerkandera adusta. The effects of the reaction conditions: VP activity, organic acid concentration and H₂O₂ addition rate, on the kinetics of the VP based oxidation system were evaluated. Diclofenac and estrogens were completely degraded after only 5–25 min even with a very low VP activity (10 U l⁻¹). High degradation percentages (80%) were achieved for sulfamethoxazole and naproxen. Low or undetectable removal yields were observed for citalopram (up to 18%), fluoxetine (lower than 10%) and carbamazepine (not degraded).     

Evidence for a one-electron mechanism of 2-aminofluorene oxidation by prostaglandin H synthase and horseradish peroxidase

Previous studies have shown that the primary arylamine carcinogen 2-aminofluorene (2-AF) is oxidized by the prostaglandin H synthase peroxidase to mutagenic and electrophilic products capable of covalent binding to macromolecules. The present study was designed to identify the potential reactive intermediate(s) responsible for binding, and to characterize further the metabolic intermediates in 2-AF peroxidation. Both prostaglandin H synthase and horseradish peroxidase, with H2O2, oxidize 2-AF to azofluorene, 2-aminodifluorenylamine (2-ADFA), 2-nitrofluorene, polymeric and nonorganic-extractable material. Both enzymes show greater activity at pH 5.0 than at pH 7.0. In the presence of either 2-t-butyl-4-methoxyphenol or 2,6-dimethylphenol, arylamine/phenol adducts were formed in high yield, with the nitrogen of either 2-AF or 2-ADFA coupled to the para position of the phenol (loss of -OCH3 with 2-t-butyl-4-methoxyphenol). These structures were confirmed by mass spectrometry and NMR spectroscopy. Acid hydrolysis of N-hydroxy-2-AF to yield the nitrenium ion, in the presence of a phenol, also results in adduct formation, but only at times greater than 2 h and in very limited yield. The peroxidase-catalyzed adduct formation, however is rapid (less than 2 min) and extensive. These and other data support a one-electron pathway for 2-AF peroxidation, with a free radical or a free radical-derived product responsible for binding to protein and DNA. An N-hydroxy intermediate may therefore not be obligatory in the enzymatic activation of 2-AF to a mutagenic product.     

Directed evolution of a temperature-, peroxide- and alkaline pH-tolerant versatile peroxidase

The VPs (versatile peroxidases) secreted by white-rot fungi are involved in the natural decay of lignin. In the present study, a fusion gene containing the VP from Pleurotus eryngii was subjected to six rounds of directed evolution, achieving a level of secretion in Saccharomyces cerevisiae (21?mg/l) as yet unseen for any ligninolytic peroxidase. The evolved variant for expression harboured four mutations and increased its total VP activity 129-fold. The signal leader processing by the STE13 protease at the Golgi compartment changed as a consequence of overexpression, retaining the additional N-terminal sequence Glu-Ala-Glu-Ala that enhanced secretion. The engineered N-terminally truncated variant displayed similar biochemical properties to those of the non-truncated counterpart in terms of kinetics, stability and spectroscopic features. Additional cycles of evolution raised the T50 8°C and significantly increased the enzyme's stability at alkaline pHs. In addition, the Km for H2O2 was enhanced up to 15-fold while the catalytic efficiency was maintained, and there was an improvement in peroxide stability (with half-lives for H2O2 of 43?min at a H2O2/enzyme molar ratio of 4000:1). Overall, the directed evolution approach described provides a set of strategies for selecting VPs with improvements in secretion, activity and stability.     

Oxidation of vacuolar and apoplastic phenolic substrates by peroxidase: Physiological significance of the oxidation reactions

Phenolic components and peroxidases are localized in vacuoles. Vacuolar peroxidase can oxidize phenolics when H₂O₂ is formed in vacuoles or tonoplasts, or when H₂O₂ formed outside of vacuoles is diffused into the organelles. In a mixture of phenolics containing a good and a poor substrate for peroxidase, a radical transfer reaction is possible from the radicals of the good substrate to the poor substrate, resulting in the enhancement of oxidation of the poor substrate. Phenoxyl radicals formed by peroxidase-dependent reactions are reduced by ascorbate in vacuoles. So, as long as ascorbate is present in vacuoles, the accumulation of oxidation products of phenolics is not significant. This suggests that ascorbate/phenolics/peroxidase systems in the vacuoles can scavenge H₂O₂. During aging, some phenolics are accumulated in vacuoles and the apoplast, and the accumulated phenolics are oxidized to brown components by peroxidase-dependent reactions. The brown components can produced O₂ ^? and H₂O₂ by autooxidation. The significance and the mechanisms of browning are discussed in tobacco leaves and onion scales.

     

Lignin transformation by a versatile peroxidase from a novel Bjerkandera sp. strain

A versatile peroxidase, purified from a novel strain of Bjerkandera sp. (B33/3), was tested for its reactivity on a lignin fraction obtained from straw pulping. The effects of such processing parameters as reaction time, pH, and lignin:enzyme ratio were evaluated. Gel filtration chromatography was employed to characterise the molecular mass distribution of the lignin fragments produced by the enzyme-mediated reaction. Our results have shown that such a versatile peroxidase can directly bring about transformations of lignin, even in the absence of external mediators.     

Molecular characterisation of a versatile peroxidase from a Bjerkandera strain

The cloning and sequencing of the rbpa gene coding for a versatile peroxidase from a novel Bjerkandera strain is hereby reported. The 1777 bp isolated fragment contained a 1698 bp peroxidase-encoding gene, interrupted by 11 introns. The 367 amino acid-deduced sequence includes a 27 amino acid-signal peptide. The molecular model, built via homology modelling with crystal structures of four fungal peroxidases, highlighted the amino acid residues putatively involved in manganese binding and aromatic substrate oxidation. The potential heme pocket residues (R44, F47, H48, E79, N85, H177, F194 and D239) include both distal and proximal histidines (H48 and H177). RBP possesses potential calcium-binding residues (D49, G67, D69, S71, S178, D195, T197, I200 and D202) and eight cysteine residues (C3, C15, C16, C35, C121, C250, C286, C316). In addition, RBP includes residues involved in substrate oxidation: three acidic residues (E37, E41 and D183)--putatively involved in manganese binding and H83 and W172--potentially involved in oxidation of aromatic substrates. Characterisation of nucleotide and amino acid sequences include RBP in versatile peroxidase group sharing catalytic properties of both LiP and MnP. In addition, the RBP enzyme appears to be closely related with the ligninolytic peroxidases from the Trametes versicolor strain.     

Competing peroxidase and oxidase reactions in scopoletin-dependent H2O2-initiated oxidation of NADH by horseradish peroxidase

Addition of NADH inhibited the peroxidative loss of scopoletin in presence of horseradish and H₂O₂ and decreased the ratio of scopoletin (consumed):H₂O₂ (added). Concomitantly NADH was oxidized and oxygen was consumed with a stoichiometry of NADH:O₂ of 2:1. On step-wise addition of a small concentration of H₂O₂ a high rate of NADH oxidation was obtained for a progressively decreasing time period followed by termination of the reaction with NADH:H₂O₂ ratio decreasing from about 40 to 10. The rate of NADH oxidation increased linearly with increase in scopoletin concentration. Other phenolic compounds including p-coumarate also supported this reaction to a variable degree. A 418-nm absorbing compound accumulated during oxidation of NADH. The effectiveness of a small concentration of H₂O₂ in supporting NADH oxidation increased in presence of SOD and decreased in presence of cytochrome c, but the reaction terminated even in their presence. The results indicate that the peroxidase is not continuously generating H₂O₂ during scopoletin-mediated NADH oxidation and that both peroxidase and oxidase reactions occur simultaneously competing for an active form of the enzyme.     

Kinetic and redox properties of MnP II, a major manganese peroxidase isoenzyme from Panus tigrinus CBS 577.79

A manganese peroxidase (MnP) isoenzyme from Panus tigrinus CBS 577.79 was produced in a benchtop stirred-tank reactor and purified to apparent homogeneity. The purification scheme involving ultrafiltration, affinity chromatography on concanavalin–A Sepharose, and gel filtration led to a purified MnP, termed “MnP II,” with a specific activity of 288 IU mg⁻¹ protein and a final yield of 22%. The enzyme turned out to be a monomeric protein with molecular mass of 50.5 kDa, pI of 4.07, and an extent of N-glycosylation of about 5.3% of the high-mannose type. The temperature and pH optima for the formation of malonate manganic chelates were 45 °C and 5.5, respectively. MnP II proved to be poorly thermostable at 50 and 60 °C, with half-lives of 11 min and 105 s, respectively. K _m values for H₂O₂ and Mn²⁺ were 16 and 124 μM, respectively. Although MnP II was able to oxidize veratryl alcohol and to catalyze the Mn²⁺-independent oxidation of several phenols, it cannot be assigned to the versatile peroxidase family. As opposed to versatile peroxidase oxidation, veratryl alcohol oxidation required the simultaneous presence of H₂O₂ and Mn²⁺; in addition, low turnover numbers and K _m values higher than 300 μM characterized the Mn²⁺-independent oxidation of substituted phenols. Kinetic properties and the substrate specificity of the enzyme markedly differed from those reported for MnP isoenzymes produced by the reference strain P. tigrinus 8/18. To our knowledge, this study reports for the first time a thorough electrochemical characterization of a MnP from this fungus.     

Transformation of halogenated pesticides by versatile peroxidase from Bjerkandera adusta

Purified versatile peroxidase (VP) from the white rot fungus Bjerkandera adusta UAMH 8258 was used to study the transformation of several pesticides, including some as highly halogenated as the wood preservative pentachlorophenol (PCP). From the 13 pesticides assayed, dichlorophen, bromoxynil and PCP were transformed by VP in the presence and in the absence of manganese(II). For all the pesticides transformed, the activity was higher in the absence of Mn(II) at pH 3 than in the presence of Mn(II) at pH 4. Catalytic constants (k_cat) in the absence of Mn(II) at pH 4 were 194 and 409 min⁻¹ for dichlorophen and bromoxynil, respectively. The K_M values were 32 and 31 μM for the pesticides and 26 and 19 μM for the hydrogen peroxide, respectively. Analysis of reaction products by GC–MS showed the presence of 2,3,5,6-tetrachloroquinone among the products from pentachlorophenol oxidation, while the main product from dichlorophen was 4-chlorophenol-2,2′-methylenequinone. Several polymers were obtained from the peroxidase oxidation of bromoxynil. In all cases, we found dehalogenation reactions mediated by the versatile peroxidase. The importance and potential uses of the enzymatic transformation of these halogenated toxic compounds is discussed.