Substrate oxidation sites in versatile peroxidase and other basidiomycete peroxidases

Versatile peroxidase (VP) is defined by its capabilities to oxidize the typical substrates of other basidiomycete peroxidases: (i) Mn(2+), the manganese peroxidase (MnP) substrate (Mn(3+) being able to oxidize phenols and initiate lipid peroxidation reactions); (ii) veratryl alcohol (VA), the typical lignin peroxidase (LiP) substrate; and (iii) simple phenols, which are the substrates of Coprinopsis cinerea peroxidase (CIP). Crystallographic, spectroscopic, directed mutagenesis, and kinetic studies showed that these 'hybrid' properties are due to the coexistence in a single protein of different catalytic sites reminiscent of those present in the other basidiomycete peroxidase families. Crystal structures of wild and recombinant VP, and kinetics of mutated variants, revealed certain differences in its Mn-oxidation site compared with MnP. These result in efficient Mn(2+) oxidation in the presence of only two of the three acidic residues forming its binding site. On the other hand, a solvent-exposed tryptophan is the catalytically-active residue in VA oxidation, initiating an electron transfer pathway to haem (two other putative pathways were discarded by mutagenesis). Formation of a tryptophanyl radical after VP activation by peroxide was detected using electron paramagnetic resonance. This was the first time that a protein radical was directly demonstrated in a ligninolytic peroxidase. In contrast with LiP, the VP catalytic tryptophan is not beta-hydroxylated under hydrogen peroxide excess. It was also shown that the tryptophan environment affected catalysis, its modification introducing some LiP properties in VP. Moreover, some phenols and dyes are oxidized by VP at the edge of the main haem access channel, as found in CIP. Finally, the biotechnological interest of VP is discussed.     

Site-directed mutagenesis of the catalytic tryptophan environment in Pleurotus eryngii versatile peroxidase

Lignin degradation by fungal peroxidases is initiated by one-electron transfer to an exposed tryptophan radical, a reaction mediated by veratryl alcohol (VA) in lignin peroxidase (LiP). Versatile peroxidase (VP) differs not only in its oxidation of Mn2+ at a second catalytic site but also in its ability to directly oxidize different aromatic compounds. The catalytic tryptophan environment was compared in LiP and VP crystal structures, and six residues near VP Trp164 were modified by site-directed mutagenesis. Oxidation of Mn2+ was practically unaffected. However, several mutations modified the oxidation kinetics of the high-redox-potential substrates VA and Reactive Black 5 (RB5), demonstrating that other residues contribute to substrate oxidation by the Trp164 radical. Introducing acidic residues at the tryptophan environment did not increase the efficiency of VP oxidizing VA. On the contrary, all variants harboring the R257D mutation lost their activity on RB5. Interestingly, this activity was restored when VA was added as a mediator, revealing the LiP-type behavior of this variant. Moreover, combination of the A260F and R257A mutations strongly increased (20-50-fold) the apparent second-order rate constants for reduction of VP compounds I and II by VA to values similar to those found in LiP. Dissociation of the enzyme-product complex seemed to be the limiting step in the turnover of this improved variant. Nonexposed residues in the vicinity of Trp164 can also affect VP activity, as found with the M247F mutation. This was a direct effect since no modification of the surrounding residues was found in the crystal structure of this variant.     

Description of a versatile peroxidase involved in the natural degradation of lignin that has both manganese peroxidase and lignin peroxidase substrate interaction sites

Two major peroxidases are secreted by the fungus Pleurotus eryngii in lignocellulose cultures. One is similar to Phanerochaete chrysosporium manganese-dependent peroxidase. The second protein (PS1), although catalyzing the oxidation of Mn2+ to Mn3+ by H2O2, differs from the above enzymes by its manganese-independent activity enabling it to oxidize substituted phenols and synthetic dyes, as well as the lignin peroxidase (LiP) substrate veratryl alcohol. This is by a mechanism similar to that reported for LiP, as evidenced by p-dimethoxybenzene oxidation yielding benzoquinone. The apparent kinetic constants showed high activity on Mn2+, but methoxyhydroquinone was the natural substrate with the highest enzyme affinity (this and other phenolic substrates are not efficiently oxidized by the P. chrysosporium peroxidases). A three-dimensional model was built using crystal models from four fungal peroxidase as templates. The model suggests high structural affinity of this versatile peroxidase with LiP but shows a putative Mn2+ binding site near the internal heme propionate, involving Glu36, Glu40, and Asp181. A specific substrate interaction site for Mn2+ is supported by kinetic data showing noncompetitive inhibition with other peroxidase substrates. Moreover, residues reported as involved in LiP interaction with veratryl alcohol and other aromatic substrates are present in peroxidase PS1 such as His82 at the heme-channel opening, which is remarkably similar to that of P. chrysosporium LiP, and Trp170 at the protein surface. These residues could be involved in two different hypothetical long range electron transfer pathways from substrate (His82-Ala83-Asn84-His47-heme and Trp170-Leu171-heme) similar to those postulated for LiP.     

Lignin peroxidase oxidation of veratryl alcohol: effects of the mutants H82A, Q222A, W171A, and F267L

The site-directed mutations H82A and Q222A (residues near the heme access channel), and W171A and F267L (residues near the surface of the protein) were introduced into the gene encoding lignin peroxidase (LiP) isozyme H8 from Phanerochaete chrysosporium. The variant enzymes were produced by homologous expression in P. chrysosporium, purified to homogeneity, and characterized by kinetic and spectroscopic methods. The molecular masses, the pIs, and the UV-vis absorption spectra of the ferric and oxidized states of these LiP variant enzymes were similar to those of wild-type LiP (wtLiP), suggesting the overall protein and heme environments were not significantly affected by these mutations. The steady-state and transient-state parameters for the oxidation of veratryl alcohol (VA) by the H82A and Q222A variants were very similar to those of wtLiP, demonstrating that these residues are not involved in VA oxidation and that the heme access channel is an unlikely site for VA oxidation. In contrast, the W171A variant was unable to oxidize VA, confirming the apparent essentiality of Trp171 in VA oxidation by LiP. The kinetic rates of spontaneous LiP compound I reduction in the absence of VA were similar for W171A and wild-type LiP, suggesting that there may not be a radical formed on the Trp171 residue of LiP in the absence of VA. For the F267L variant, both the K(m app) value in the steady state and the apparent dissociation constant (K(D)) for compound II reduction were greater than those for wtLiP. These results indicate that the site including W171 and F267, rather than the heme access channel, is the site of VA binding and oxidation in LiP. Whereas Trp171 appears to be essential for VA oxidation, it apparently is not independently responsible for the spontaneous decomposition of oxidized intermediates. The nearby Phe267 apparently is also involved in VA binding.     

The crystal structure of lignin peroxidase at 1.70 A resolution reveals a hydroxy group on the cbeta of tryptophan 171: a novel radical site formed during the redox cycle

The crystal structure of lignin peroxidase (LiP) from the white rot fungus Phanerochaete chrysosporium was refined to an R-factor of 16.2 % utilizing synchrotron data in the resolution range from 10 to 1.7 A. The final model comprises all 343 amino acid residues, 370 water molecules, the heme, four carbohydrates, and two calcium ions. Lignin peroxidase shows the typical peroxidase fold and the heme has a close environment as found in other peroxidases. During refinement of the LiP model an unprecedented modification of an amino acid was recognized. The surface residue tryptophan 171 in LiP is stereospecifically hydroxylated at the Cbeta atom due to an autocatalytic process. We propose that during the catalytic cycle of LiP a transient radical at Trp171 occurs that is different from those previously assumed for this type of peroxidase. Recently, the existence of a second substrate-binding site centered at Trp171 has been reported, by us which is different from the "classical heme edge" site found in other peroxidases. Here, we report evidence for a radical formation at Trp171 using spin trapping, which supports the concept of Trp171 being a redox active amino acid and being involved in the oxidation of veratryl alcohol. On the basis of our current model, an electron pathway from Trp171 to the heme is envisaged, relevant for the oxidation of veratryl alcohol and possibly lignin. Beside the opening leading to the heme edge, which can accommodate small aromatic substrate molecules, a smaller channel giving access to the distal heme pocket was identified that is large enough for molecules such as hydrogen peroxide. Furthermore, it was found that in LiP the bond between the heme iron and the Nepsilon2 atom of the proximal histidine residue is significantly longer than in cytochrome c peroxidase (CcP). The weaker Fe-N bond in LiP renders the heme more electron deficient and destabilizes high oxidation states, which could explain the higher redox potential of LiP as compared to CcP.     

First synthesis of a polysaccharide-supported lignin model compound and study of its oxidation promoted by lignin peroxidase

Veratrylchitosan, a polysaccharide-supported lignin model compound, has been synthesised by covalently attaching 3-(3,4-dimethoxybenzyloxy)propionic acid to the polysaccharide chitosan through an amide linkage. When this polymer was used as a substrate in the oxidation promoted by lignin peroxidase (LiP), significant decomposition of the lignin model resulted in the formation of veratraldehyde. The oxidation mechanism involves an initial transfer of one electron from chitosan to the active species of LiP (LiP I) followed by C(alpha)-H deprotonation of an aromatic cation radical. A benzylic radical is then formed which is further oxidised to a benzyl cation. Reaction with water and hydrolysis of the hemiacetal then lead to veratraldehyde formation. An increase in the yields of the oxidation product is observed in the presence of the mediator 2-chloro-1,4-dimethoxybenzene, thus indicating that a more efficient degradation results from the transfer of an electron from the polymer to the radical cation of the mediator.     

Oxidative polymerization of ribonuclease A by lignin peroxidase from Phanerochaete chrysosporium. Role of veratryl alcohol in polymer oxidation.

The mechanism of lignin peroxidase (LiP) was examined using bovine pancreatic ribonuclease A (RNase) as a polymeric lignin model substrate. SDS/PAGE analysis demonstrates that an RNase dimer is the major product of the LiP-catalyzed oxidation of this protein. Fluorescence spectroscopy and amino acid analyses indicate that RNase dimer formation is due to the LiP-catalyzed oxidation of Tyr residues to Tyr radicals, followed by intermolecular radical coupling. The LiP-catalyzed polymerization of RNase in strictly dependent on the presence of veratryl alcohol (VA). In the presence of 100 microM H2O2, relatively low concentrations of RNase and VA, together but not individually, can protect LiP from H2O2 inactivation. The presence of RNase strongly inhibits VA oxidation to veratraldehyde by LiP; whereas the presence of VA does not inhibit RNase oxidation by LiP. Stopped-flow and rapid-scan spectroscopy demonstrate that the reduction of LiP compound I (LiPI) to the native enzyme by RNase occurs via two single-electron steps. At pH 3.0, the reduction of LiPI by RNase obeys second-order kinetics with a rate constant of 4.7 x 10(4) M-1.s-1, compared to the second-order VA oxidation rate constant of 3.7 x 10(5) M-1.s-1. The reduction of LiP compound II (LiPII) by RNase also follows second-order kinetics with a rate constant of 1.1 x 10(4) M-1.s-1, compared to the first-order rate constant for LiPII reduction by VA. When the reductions of LiPI and LiPIi are conducted in the presence of both VA and RNase, the rate constants are essentially identical to those obtained with VA alone. These results suggest that VA is oxidized by LiP to its cation radical which, while still in its binding site, oxidizes RNase.     

Tryptophan-based radical in the catalytic mechanism of versatile peroxidase from Bjerkandera adusta

Versatile peroxidase (VP) from Bjerkandera adusta is a structural hybrid between lignin (LiP) and manganese (MnP) peroxidase. This hybrid combines the catalytic properties of the two above peroxidases, being able to oxidize typical LiP and MnP substrates. The catalytic mechanism is that of classical peroxidases, where the substrate oxidation is carried out by a two-electron multistep reaction at the expense of hydrogen peroxide. Elucidation of the structures of intermediates in this process is crucial for understanding the mechanism of substrate oxidation. In this work, the reaction of H(2)O(2) with the enzyme in the absence of substrate has been investigated with electron paramagnetic resonance (EPR) spectroscopy. The results reveal an EPR signal with partially resolved hyperfine structure typical of an organic radical. The yield of this radical is approximately 30%. Progressive microwave power saturation measurements indicate that the radical is weakly coupled to a paramagnetic metal ion, suggesting an amino acid radical in moderate distance from the ferryl heme. A tryptophan radical was identified as a protein-based radical formed during the catalytic mechanism of VP from Bjerkandera adusta through X-band and high-field EPR measurements at 94 GHz, aided by computer simulations for both frequency bands. A close analysis of the theoretical model of the VP from Bjerkandera sp. shows the presence of a tryptophan residue near to the heme prosthetic group, which is solvent-exposed as in the case of LiP and other VPs. The catalytic role of this residue in a long-range electron-transfer pathway is discussed.     

Demonstration of Lignin-to-Peroxidase Direct Electron Transfer: A TRANSIENT-STATE KINETICS,DIRECTED MUTAGENESIS,EPR, AND NMR STUDY*

Versatile peroxidase (VP) is a high redox-potential peroxidase of biotechnological interest that is able to oxidize phenolic and non-phenolic aromatics, Mn²⁺, and different dyes. The ability of VP from Pleurotus eryngii to oxidize water-soluble lignins (softwood and hardwood lignosulfonates) is demonstrated here by a combination of directed mutagenesis and spectroscopic techniques, among others. In addition, direct electron transfer between the peroxidase and the lignin macromolecule was kinetically characterized using stopped-flow spectrophotometry. VP variants were used to show that this reaction strongly depends on the presence of a solvent-exposed tryptophan residue (Trp-164). Moreover, the tryptophanyl radical detected by EPR spectroscopy of H₂O₂-activated VP (being absent from the W164S variant) was identified as catalytically active because it was reduced during lignosulfonate oxidation, resulting in the appearance of a lignin radical. The decrease of lignin fluorescence (excitation at 355 nm/emission at 400 nm) during VP treatment under steady-state conditions was accompanied by a decrease of the lignin (aromatic nuclei and side chains) signals in one-dimensional and two-dimensional NMR spectra, confirming the ligninolytic capabilities of the enzyme. Simultaneously, size-exclusion chromatography showed an increase of the molecular mass of the modified residual lignin, especially for the (low molecular mass) hardwood lignosulfonate, revealing that the oxidation products tend to recondense during the VP treatment. Finally, mutagenesis of selected residues neighboring Trp-164 resulted in improved apparent second-order rate constants for lignosulfonate reactions, revealing that changes in its protein environment (modifying the net negative charge and/or substrate accessibility/binding) can modulate the reactivity of the catalytic tryptophan.     

Catalytic mechanisms and regulation of lignin peroxidase.

Lignin peroxidase (LiP) is a fungal haemoprotein similar to the lignin-synthesizing plant peroxidases, but it has a higher oxidation potential and oxidizes dimethoxylated aromatic compounds to radical cations. It catalyses the degradation of lignin models but in vitro the outcome is net lignin polymerization. LiP oxidizes veratryl alcohol to radical cations which are proposed to act by charge transfer to mediate in the oxidation of lignin. Phenolic compounds are, however, preferentially oxidized, but transiently inactivate the enzyme. Analysis of the catalytic cycle of LiP shows that in the presence of veratryl alcohol the steady-state turnover intermediate is Compound II. We propose that veratryl alcohol is oxidized by the enzyme intermediate Compound I to a radical cation which now participates in charge-transfer reactions with either veratryl alcohol or another reductant, when present. Reduction of Compound II to native state may involve a radical product of veratryl alcohol or radical product of charge transfer. Phenoxy radicals, by contrast, cannot engage in charge-transfer reactions and reaction of Compound II with H2O2 ensues to form the peroxidatically inactive intermediate, Compound III. Regulation of LiP activity by phenolic compounds suggests feedback control, since many of the products of lignin degradation are phenolic. Such control would lower the concentration of phenolics relative to oxygen and favour degradative ring-opening reactions.     

Degradation of azo compounds by ligninase from Phanerochaete chrysosporium: involvement of veratryl alcohol

Phanerochaete chrysosporium decolorized several polyaromatic azo dyes in ligninolytic culture. The oxidation rates of individual dyes depended on their structures. Veratryl alcohol stimulated azo dye oxidation by pure lignin peroxidase (ligninase, LiP) in vitro. Accumulation of compound II of lignin peroxidase, an oxidized form of the enzyme, was observed after short incubations with these azo substrates. When veratryl alcohol was also present, only the native form of lignin peroxidase was observed. Azo dyes acted as inhibitors of veratryl alcohol oxidation. After an azo dye had been degraded, the oxidation rates of veratryl alcohol recovered, confirming that these two compounds competed for ligninase during the catalytic cycle. Veratryl alcohol acts as a third substrate (with H2O2 and the azo dye) in the lignin peroxidase cycle during oxidations of azo dyes.     

Determination of a catalytic tyrosine in <Emphasis Type="Italic">Trametes cervina</Emphasis> lignin peroxidase with chemical modification techniques

Trametes cervina lignin peroxidase (LiP) lacks a catalytic tryptophan strictly conserved in other LiP and versatile peroxidases. It contains tyrosine¹⁸¹ at the potential catalytic site. This protein and the well-characterized Phanerochaete chrysosporium LiP with the catalytic tryptophan¹⁷¹ have been chemically modified: the tryptophan-specific modification with N-bromosuccinimide sufficiently disrupted oxidation of veratryl alcohol by P. chrysosporium LiP, whereas the activity of T. cervina LiP was not affected, suggesting no catalytic tryptophan in T. cervina LiP. On the other hand, the tyrosine-specific modification with tetranitromethane did not affect the activities of P. chrysosporium LiP lacking tyrosine but inactivated T. cervina LiP due to the nitration of tyrosine¹⁸¹. These results strongly suggest that tyrosine¹⁸¹ is at the catalytic site in T. cervina LiP.     

Lignin-degrading peroxidases from genome of selective ligninolytic fungus Ceriporiopsis subvermispora

The white-rot fungus Ceriporiopsis subvermispora delignifies lignocellulose with high selectivity, but until now it has appeared to lack the specialized peroxidases, termed lignin peroxidases (LiPs) and versatile peroxidases (VPs), that are generally thought important for ligninolysis. We screened the recently sequenced C. subvermispora genome for genes that encode peroxidases with a potential ligninolytic role. A total of 26 peroxidase genes was apparent after a structural-functional classification based on homology modeling and a search for diagnostic catalytic amino acid residues. In addition to revealing the presence of nine heme-thiolate peroxidase superfamily members and the unexpected absence of the dye-decolorizing peroxidase superfamily, the search showed that the C. subvermispora genome encodes 16 class II enzymes in the plant-fungal-bacterial peroxidase superfamily, where LiPs and VPs are classified. The 16 encoded enzymes include 13 putative manganese peroxidases and one generic peroxidase but most notably two peroxidases containing the catalytic tryptophan characteristic of LiPs and VPs. We expressed these two enzymes in Escherichia coli and determined their substrate specificities on typical LiP/VP substrates, including nonphenolic lignin model monomers and dimers, as well as synthetic lignin. The results show that the two newly discovered C. subvermispora peroxidases are functionally competent LiPs and also suggest that they are phylogenetically and catalytically intermediate between classical LiPs and VPs. These results offer new insight into selective lignin degradation by C. subvermispora.     

Mediator role of veratryl alcohol in the lignin peroxidase-catalyzed oxidative decolorization of Remazol Brilliant Blue R

Lignin peroxidase (LiP) produced by Trametes versicolor decolorizes Remazol Brilliant Blue R (RBBR) in the presence as well as in the absence of veratryl alcohol (VA). VA enhances and stabilizes the RBBR-decolorization rates by lignin peroxidase. RBBR has better substrate reactivity than VA for LiP. RBBR is also decolorized directly by LiP and competitively inhibits VA oxidation by LiP. In the presence of higher concentrations of RBBR (i) RBBR decolorization rates improve, (ii) veratryl aldehyde appears after a lag and (iii) VA oxidation rates decrease. The lag is due to consumption of VA cation radical (VA⁺) generated upon LiP-catalyzed VA oxidation, during RBBR oxidation. That may result in the formation of compound III in the absence of VA⁺ and contributes to the inhibitory influence of RBBR on LiP activity.     

Kinetic and thermodynamic characterization of the functional properties of a hybrid versatile peroxidase using isothermal titration calorimetry: Insight into manganese peroxidase activation and lignin peroxidase inhibition

Isothermal titration calorimetry (ITC) was developed for measuring lignin peroxidase (LiP) and manganese peroxidase (MnP) activities of versatile peroxidase (VP) from Bjerkandera adusta. Developing an ITC approach provided an alternative to colorimetric methods that enabled reaction kinetics to be accurately determined. Although VP from Bjerkandera adjusta is a hybrid enzyme, specific conditions of [Mn+2] and pH were defined that limited activity to either LiP or MnP activities, or enabled both to be active simultaneously. MnP activity was found to be more efficient than LiP activity, with activity increasing with increasing concentrations of Mn+2. These properties of MnP were explained by a second metal binding site involved in homotropic substrate (Mn+2) activation. The activation of MnP was also accompanied by a decrease in both activation energy and substrate (Mn) affinity, reflecting a flexible enzyme structure. In contrast to MnP activity, LiP activity was inhibited by high dye (substrate) concentrations arising from uncompetitive substrate inhibition caused by substrate binding to a site distinct from the catalytic site. Our study provides a new level of understanding about the mechanism of substrate regulation of catalysis in VP from B. adjusta, providing insight into a class of enzyme, hybrid class II peroxidases, for which little experimental data is available.     

A novel enzymatic decarboxylation of oxalic acid by the lignin peroxidase system of white-rot fungus Phanerochaete chrysosporium

Oxidation of veratryl alcohol by lignin peroxidase (LiP) was potently inhibited by oxalic acid. The inhibition analysis with Lineweaver-Burk plots clearly showed that the type of inhibition is non-competitive. The enzymatic oxidation of veratryl alcohol in the presence of 14C-oxalic acid yielded radioactive carbon dioxide. The results indicate that the apparent inhibition of LiP is caused by reduction of the veratryl alcohol cation radical intermediate back to the substrate level by oxalate, which is concomitantly oxidized to carbon dioxide.     

Two Oxidation Sites for Low Redox Potential Substrates: A DIRECTED MUTAGENESIS,KINETIC, AND CRYSTALLOGRAPHIC STUDY ON PLEUROTUS ERYNGII VERSATILE PEROXIDASE*

Versatile peroxidase shares with manganese peroxidase and lignin peroxidase the ability to oxidize Mn²⁺ and high redox potential aromatic compounds, respectively. Moreover, it is also able to oxidize phenols (and low redox potential dyes) at two catalytic sites, as shown by biphasic kinetics. A high efficiency site (with 2,6-dimethoxyphenol and p-hydroquinone catalytic efficiencies of ∼70 and ∼700 s⁻¹ mm⁻¹, respectively) was localized at the same exposed Trp-164 responsible for high redox potential substrate oxidation (as shown by activity loss in the W164S variant). The second site, characterized by low catalytic efficiency (∼3 and ∼50 s⁻¹ mm⁻¹ for 2,6-dimethoxyphenol and p-hydroquinone, respectively) was localized at the main heme access channel. Steady-state and transient-state kinetics for oxidation of phenols and dyes at the latter site were improved when side chains of residues forming the heme channel edge were removed in single and multiple variants. Among them, the E140G/K176G, E140G/P141G/K176G, and E140G/W164S/K176G variants attained catalytic efficiencies for oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) at the heme channel similar to those of the exposed tryptophan site. The heme channel enlargement shown by x-ray diffraction of the E140G, P141G, K176G, and E140G/K176G variants would allow a better substrate accommodation near the heme, as revealed by the up to 26-fold lower K_m values (compared with native VP). The resulting interactions were shown by the x-ray structure of the E140G-guaiacol complex, which includes two H-bonds of the substrate with Arg-43 and Pro-139 in the distal heme pocket (at the end of the heme channel) and several hydrophobic interactions with other residues and the heme cofactor.     

Understanding lignin-degrading reactions of ligninolytic enzymes: binding affinity and interactional profile

Previous works have demonstrated that ligninolytic enzymes mediated effective degradation of lignin wastes. The degrading ability greatly relied on the interactions of ligninolytic enzymes with lignin. Ligninolytic enzymes mainly contain laccase (Lac), lignin peroxidase (LiP) and manganese peroxidase (MnP). In the present study, the binding modes of lignin to Lac, LiP and MnP were systematically determined, respectively. Robustness of these modes was further verified by molecular dynamics (MD) simulations. Residues GLU460, PRO346 and SER113 in Lac, residues ARG43, ALA180 and ASP183 in LiP and residues ARG42, HIS173 and ARG177 in MnP were most crucial in binding of lignin, respectively. Interactional analyses showed hydrophobic contacts were most abundant, playing an important role in the determination of substrate specificity. This information is an important contribution to the details of enzyme-catalyzed reactions in the process of lignin biodegradation, which can be used as references for designing enzyme mutants with a better lignin-degrading activity.     

The cloning of a new peroxidase found in lignocellulose cultures of Pleurotus eryngii and sequence comparison with other fungal peroxidases

We report cloning and sequencing of gene ps1 encoding a versatile peroxidase combining catalytic properties of lignin peroxidase (LiP) and manganese peroxidase (MnP) isolated from lignocellulose cultures of the white-rot fungus Pleurotus eryngii. The gene contains 15 putative introns, and the deduced amino acid sequence consists of a 339-residue mature protein with a 31-residue signal peptide. Several putative response elements were identified in the promoter region. Amino acid residues involved in oxidation of Mn(2+) and aromatic substrates by direct electron transfer to heme and long-range electron transfer from superficial residues as predicted by analogy with Phanerochaete chrysosporium MnP and LiP, respectively. A dendrogram is presented illustrating sequence relationships between 29 fungal peroxidases.     

Lignin peroxidase oxidation of Mn2+ in the presence of veratryl alcohol, malonic or oxalic acid, and oxygen

Veratryl alcohol (3,4-dimethoxybenzyl alcohol) appears to have multiple roles in lignin degradation by Phanerochaete chrysosporium. It is synthesized de novo by the fungus. It apparently induces expression of lignin peroxidase (LiP), and it protects LiP from inactivation by H2O2. In addition, veratryl alcohol has been shown to potentiate LiP oxidation of compounds that are not good LiP substrates. We have now observed the formation of Mn3+ in reaction mixtures containing LiP, Mn2+, veratryl alcohol, malonate buffer, H2O2, and O2. No Mn3+ was formed if veratryl alcohol or H2O2 was omitted. Mn3+ formation also showed an absolute requirement for oxygen, and oxygen consumption was observed in the reactions. This suggests involvement of active oxygen species. In experiments using oxalate (a metabolite of P. chrysosporium) instead of malonate, similar results were obtained. However, in this case, we detected (by ESR spin-trapping) the production of carbon dioxide anion radical (CO2.-) and perhydroxyl radical (.OOH) in reaction mixtures containing LiP, oxalate, veratryl alcohol, H2O2, and O2. Our data indicate the formation of oxalate radical, which decays to CO2 and CO2.-. The latter reacts with O2 to form O2.-, which then oxidizes Mn2+ to Mn3+. No radicals were detected in the absence of veratryl alcohol. These results indicate that LiP can indirectly oxidize Mn2+ and that veratryl alcohol is probably a radical mediator in this system.