VPs (versatile peroxidases) sharing the functions of LiP (lignin peroxidase) and MnP (manganese peroxidase) have been described in basidiomycetous fungi Pleurotus and Bjerkandera. Despite the importance of this enzyme in polymer degradation, its reactivity with polymeric substrates remains poorly understood. In the present study, we first report that, unlike LiP, VP from Pleurotus ostreatus directly oxidized two polymeric substrates, bovine pancreatic RNase and Poly R-478, through a long-range electron pathway without redox mediators. P. ostreatus produces several MnP isoenzymes, including the multifunctional enzyme MnP2 (VP) and a typical MnP isoenzyme MnP3. MnP2 (VP) depolymerized a polymeric azo dye, Poly R-478, to complete its catalytic cycle. Reduction of the oxidized intermediates of MnP2 (VP) to its resting state was also observed for RNase. RNase inhibited the oxidation of VA (veratryl alcohol) in a competitive manner. Blocking of the exposed tryptophan by N-bromosuccinimide inhibited the oxidation of RNase and VA by MnP2 (VP), but its Mn2+-oxidizing activity was retained, suggesting that Trp-170 exposed on an enzyme surface is a substrate-binding site both for VA and the polymeric substrates. The direct oxidation of RNase and Poly R by MnP2 (VP) is in sharp contrast with redox mediator-dependent oxidation of these polymers by LiP from Phanerochaete chrysosporium. Molecular modelling of MnP2 (VP) revealed that the differences in the dependence on redox mediators in polymer oxidation by MnP2 (VP) and LiP were explained by the anionic microenvironment surrounding the exposed tryptophan.
White rot fungi are capable of degrading oxidatively the heterogeneous aromatic polymer lignin. Pivotal extracellular enzymes of the fungal ligninolytic system are laccase (Lac) and two haemcontaining peroxidases, LiP (lignin peroxidase) and MnP (manganese peroxidase). The fourth ligninolytic enzyme is a peroxidase sharing the catalytic properties of LiP and MnP. The multifunctional enzymes were designated VPs (versatile peroxidases) and have been described in the genera Pleurotus and Bjerkandera. The multifunctional enzymes, VPs, have been isolated from cultures of Pleurotus eryngii , P. pulmonarius , P. sajor-caju  and P. ostreatus [4,5]. At present no crystal structure is available for VPs. However, molecular models built using LiP, MnP and other peroxidases as templates show the presence of a manganese-binding site in the proximity of the haem ring, on the side of the two haem propionate groups, in a manner similar to that found in MnP . The multifunctional enzymes also possess an exposed tryptophan residue, which functions as a redox active site for VA (veratryl alcohol) oxidation  as found in LiP. VPs oxidize Mn2+ to Mn3+, VA, the non-phenolic lignin dimer veratryl glycerol-β-guaiacyl ether, p-dimethoxybenzene, quinols and substituted phenols. The existence of different substrate oxidation sites in P. eryngii VPs is supported by non-competitive inhibition between oxidation of Mn2+ and high redox-potential dyes [8–10].
Recently, we reported differential transcriptional regulation of MnP isoenzymes, MnP2 and MnP3, on PGY medium (peptone–glucose–yeast extract medium) and GY medium (glucose–yeast extract medium) by P. ostreatus . Although P. ostreatus MnP2 has been classified as an MnP isoenzyme due to its manganese-oxidizing activity, analysis of catalytic functions and nucleotide sequence revealed that MnP2 is classified as a typical VP having an exposed tryptophan (Trp-170) residue which functions as a redox active site for VA oxidation. MnP2 (VP) from P. ostreatus exhibits 95.8% homology in amino acid sequence with VP from P. eryngii (PS1). pI and the molecular mass of MnP2 (VP) were 3.77 and 42 kDa respectively. As reported for VPs from P. eringii, P. ostreatus MnP2 (VP) oxidized Mn2+ and VA, at optimum pH values 5.0 and 3.0 respectively. However, the Km value of MnP2 (VP) for VA (646 μM)  was smaller than those reported for P. eringii VPs (3000–3500 μM) [1,7].
LiP is capable of oxidizing polymeric substrates. The haem in LiP is buried, ruling out a direct interaction between the haem and the polymeric substrates. Therefore the involvement of long-range electron transfer from the enzyme surface to the haem has been proposed . A previous study on an MnP variant, rMnP S168W, suggests that the exposed tryptophan on the enzyme surface plays a central role in the polymer oxidation , but the role of Trp-171 of LiP-H8 in the polymer oxidation is not fully understood. Sheng and Gold  showed that bovine pancreatic RNase was oxidized by LiP. The oxidation was strictly dependent on the presence of VA, supporting the involvement of the VA–LiP cation radical complex in the polymer oxidation. The oxidation of a polymeric dye, Poly R-478, was also catalysed by LiP in the presence of redox mediators such as VA, 1,4-dimethoxybenzene and 2-chloro-1,4-dimethoxybenzene . In contrast, ferrocytochrome c was oxidized by LiP in the absence of redox mediators . Although VP shares the molecular structure of LiP and MnP, its catalytic functions in polymer oxidation have not been analysed in detail.
In the present study, we report the catalytic functions of P. ostraeatus MnP2 (VP) in polymer oxidation. We herein first show that compounds I and II of the multifunctional enzyme MnP2 (VP) were directly reduced by the polymeric substrates RNase and Poly R-478, without the aid of redox mediators. We also discuss the differences in polymer oxidation by MnP2 (VP) and LiP based on the molecular modelling of these enzymes.
MATERIALS AND METHODS
Fungal cultures and enzyme purification
P. ostreatus A.T.C.C. 66376 was cultivated on (i) GY medium containing 1% glucose, 1% Kirk's salt solution, 0.02% yeast extract, 0.27 mM MnSO4 and 20 mM Na-phthalate buffer (pH 4.5) and (ii) PGY medium with 0.5% peptone in GY as reported in . MnP2 (VP) was isolated from GY using Sepharose CL-6B (Amersham Biosciences, Piscataway, U.S.A.) and Pharmacia Mono-Q (10/10) (Amersham Biosciences) columns. MnP3 was isolated from PGY medium using Sepharose CL-6B, Superdex 75 pg (Amersham Biosciences) and Mono-Q (10/10) columns . MnP activity was assayed using 0.4 mM guaiacol as a substrate. One unit of MnP was defined as the amount of enzyme that increased the A465 by 1.0 min−1 . The two manganese oxidizing enzymes, MnP2 and MnP3, were purified as reported in . Specific activity of MnP2 and MnP3 was 369 and 331 units/mg respectively. The genomic sequence analysis of the enzymes indicated that the MnP2 and MnP3 genes are identical with the previously cloned P. ostreatus mnp2 expressed in a sawdust culture  and mnp3 expressed in a liquid culture  respectively. The number of introns and their positions in the genes were also identical  but the nucleotide sequence of the intron in the P. ostreatus mnp2 and mnp3 genes was different . MnP2 and MnP3 isoenzymes described in the present study are the same as those designated MnP-GY and MnP-PGY in the previous report . MnP2 is a VP that is capable of oxidizing VA. MnP3 has no redox active tryptophan exposed on the enzyme surface [7,11].
Decolourization of Poly R-478
Reactions of Poly R-478 (Sigma–Aldrich, St. Louis, MO, U.S.A.) with MnPs were performed in 0.02% Poly R-478, 0.1 mM of MnSO4, 2.0 units of MnP and 50 mM sodium lactate buffer (pH 4.5). The reaction was initiated by the addition of 0.1 mM H2O2. H2O2 was added stepwise for 30 min. The final concentration of H2O2 was 0.5 mM. The reaction mixture was incubated at 28 °C for 24 h in darkness. Decolourization of Poly R-478 was assayed at A520/350. In the experiments of Poly R-478 decolourization, sodium lactate buffer was used to stabilize manganese ions formed.
Analysis of molecular mass distribution
The molecular mass distribution of Poly R-478 after the decolourization experiments was analysed by GPC (gel-permeation chromatography) on a Shodex SB803HQ column (Showa Denko, Tokyo, Japan). Elution was performed with a 0.02% sodium azide solution at a flow rate of 0.5 ml/min using a Hitachi L-7100 HPLC system (Tokyo, Japan) connected to an L4500 Diode-array detector. Weight average (Mw) and number average (Mn) molecular masses were estimated using VA (Mw 0.168 kDa) and a standard made from sodium salts of polystyrene sulphonic acid of Mw 1.4, 13 and 49 kDa (Sigma–Aldrich).
Reduction of oxidized MnP by polymeric substrates
Compound I of MnP was prepared by the addition of 1 mol of H2O2 to MnP in the resting state. Compound II of MnP was prepared by the addition of 1 eq. of H2O2 to compound I. Reduction of the oxidized form of MnP was assayed in a solution (50 μl) containing MnP (2.0 units), Poly R-478 (3.5 μg/50 μl) or RNase (40 μg/50 μl) in 50 mM sodium succinate buffer (pH 4.5) at 25 °C using a UV–VIS spectrophotometer U-3500 (Hitachi). In this experiment, sodium succinate buffer was used because the reaction mixture contained no manganese ions.
Reactions of RNase
Oxidation of RNase (Mw 13.7 kDa) from bovine pancreas (Sigma–Aldrich) was performed by adding 60 μM H2O2 to a 50 mM sodium tartrate buffer solution (pH 3.0, 3.0 ml) containing 8 μg/ml MnP and 10 μM RNase at 25 °C. Reactions of free tyrosine were performed using 600 μM tyrosine instead of RNase. Fluorescence due to coupling of the tyrosine residues was monitored by a fluorescence meter (Shimadzu, Kyoto, Japan) with an excitation wavelength of 315 nm. Emission spectra were recorded at 325–500 nm.
To detect oxidized products of RNase by SDS/PAGE, RNase was reacted with MnP2 (VP) for 24 h. The reaction was initiated by adding 60 μM H2O2 to a 50 mM sodium tartrate buffer solution (pH 3.0, 3.0 ml) containing 8 μg/ml MnP and 10 μM RNase at 25 °C. The reaction mixture was concentrated by ultrafiltration using an Amicon-10 membrane. The concentrate was subjected to SDS/PAGE using a FastGel gradient 10–15 (Amersham Biosciences). The gel was stained with Coomassie Brilliant Blue R-250.
Steady state kinetic studies of native MnP2 (VP)
Effects of Poly R-478 on the oxidation of Mn2+ by MnP2 (VP) were examined by adding 100 μM H2O2 to a 50 mM sodium tartrate buffer solution (pH 3.0) containing 0.5 μg/ml MnP2 (VP), 5.0–200 μM Mn2+ and 100 μM H2O2 with and without 0.0025% Poly R-478. The production of Mn3+ was assayed by measuring A238 using a U-2001 UV–VIS spectrophotometer (Hitachi) at 25 °C. The total volume of the reaction mixture was 1.0 ml.
Effects of RNase on the oxidation of VA by MnP2 (VP) were examined by adding 100 μM H2O2 to a 50 mM sodium tartrate buffer solution (pH 3.0) containing 5 μg/ml MnP2 (VP), 50–1200 μM VA and 0, 0.8 and 3.0 μM RNase. The oxidation of VA was assayed by measuring A310.
Steady state kinetic studies of chemically modified MnP2 (VP)
NBS (N-bromosuccinimide) was purified by recrystallization from water. NBS-MnP2 (VP) was prepared as reported for the chemical modification of the tryptophan residue in LiP . MnP2 (VP) (3.0 μM) was dissolved in 50 mM sodium acetate buffer (pH 4.0). To this solution, NBS (20 or 40 μM) was added and allowed to react at room temperature (25 °C). After 30 min, the solution of NBS-MnP2 (VP) was dialysed against 20 mM sodium succinate buffer (pH 4.5) and concentrated by ultrafiltration. The concentration of the chemically modified enzyme was calculated from the absorption maximum of the Soret region at 407 nm. Spectral changes in the Soret region were not observed after the chemical modification.
Oxidation of VA and Poly R-478 by wild-type and chemically modified MnP2 (VP) was assayed in a 50 mM sodium tartrate buffer solution (pH 3.0) containing 100 μM H2O2, substrates (1.6 mM VA or Poly R-478, 0.0125%, w/v) and the enzyme (5 μg/ml). Oxidation of Mn2+ was assayed in a 50 mM sodium tartrate buffer (pH 4.5) containing 100 μM H2O2 and 0.5 mM Mn2+ and the enzyme (5 μg/ml).
Molecular modelling was performed with a WebLab Viewer. The models of MnP2 (VP) and MnP3 were built by homology modelling (CAFASP3 server) with ESyPred3D using an amino acid sequence deduced from the genomic sequences of the enzymes .
Decolourization rate of Poly R-478 by MnP isoenzyme
MnP2 (VP) was allowed to react with Poly R-478. As shown in Table 1, MnP2 (VP) decreased A520/350 by 42.6%, in the presence of Mn2+ and H2O2. However, this enzyme decomposed the dye to decrease the A520/350 by 49.7% in the absence of Mn2+. Thus it was found that MnP2 (VP) is capable of decolourizing Poly R-478, regardless of the presence of Mn2+. MnP3 decolourized Poly R-478 by 39% on the addition of Mn2+ and H2O2. However, decolourization was not observed in the absence of Mn2+ and H2O2 (results not shown). To exclude the possibility that the decolourization of the dyes was due to abiotic oxidation with H2O2, the dye was incubated with 0.1 mM H2O2 in the absence of enzyme at 25 °C for 24 h. No spectral changes of the dye were observed in the control experiments. Thus MnP2 (VP) and MnP3 showed striking differences in Poly R decolourization.
Depolymerization of Poly-R 478 by MnP isoenzymes
The molecular weight distribution of Poly R-478 was analysed by GPC (Figure 1). The GPC profile showed that Poly R-478 with Mw 40000 was intensively depolymerized by MnP2 (VP) to lower molecular mass fragments. The GPC profile of the dye obtained from the reactions without Mn2+ was the same as that with Mn2+. Thus it was found that MnP2 (VP) is capable of decomposing Poly R-478 without the involvement of the redox cycle of manganese ions. In the presence of H2O2, depolymerization of Poly R-478 by MnP3 was observed, but the decomposition of the dye into fragments of <10000 was not observed (results not shown). Differences in the dependence of Mn2+ on the depolymerization of the dye by MnP2 (VP) and MnP3 coincided with the results of decolourization experiments.
Gel permeation chromatograms of Poly R-478 degraded by MnP2 (VP)
Reduction of oxidized intermediates of MnP isoenzymes by Poly R-478
The reduction of oxidized intermediates of MnP isoenzymes by Poly R was analysed by measuring UV–VIS spectra of the enzymes every minute. Electron absorption maxima in MnP2 (VP) were 407, 502 and 637 nm for the resting enzyme, 407, 532 and 647 nm for compound I, and 418, 529 and 558 nm for compound II. Compound I of MnP2 (VP) was formed on addition of 1 mol of H2O2. Addition of Poly R-478 to the reaction mixture changed the spectrum of the enzyme from compound I to the resting state (Figure 2). Without Poly R-478, compound I of the enzyme was stable for over 10 min. Thus it was found that oxidized intermediates of MnP2 (VP) were reduced directly by the polymer to complete its catalytic cycle. Compounds I and II of MnP3 were not reduced by Poly R-478 (results not shown).
Reduction of the MnP2 (VP) compound I by Poly R-478
MnP2 (VP)-catalysed polymerization of RNase
The MnP2 (VP)-catalysed oxidation of RNase was analysed by fluorescence spectroscopy as described in (Figure 3). It was reported that the fluorescence spectra obtained by the oxidation of RNase by LiP were identical with those reported for peroxidase-catalysed dityrosine . In the present study, we reacted free tyrosine with MnP2 (VP) and confirmed that dityrosin exhibited spectral characteristics similar to those reported before. In the reaction of RNase with LiP, the formation of a dimeric compound was strictly dependent on the presence of a redox mediator, VA .
Fluorescence spectra of RNase reacted with MnP2 (VP)
In sharp contrast with the reactions by LiP , the reaction of RNase with P. ostreatus MnP2 (VP) produced dityrosine in the absence of a redox mediator. SDS/PAGE showed that the dimer was produced with concomitant formation of a trace amount of a trimer, depending on the presence of the enzyme and H2O2 (Figure 4).
SDS/PAGE of oxidation products of RNase by MnP2 (VP)
The reduction of oxidized intermediates of MnP2 (VP) by RNase was analysed by measuring UV–VIS spectra of the enzymes. Addition of RNase to the reaction mixture changed the spectrum of the enzyme from compound I to the resting state (Figure 5). Without RNase, compound I of the enzyme was stable for over 10 min. Thus it was found that oxidized intermediates of MnP2 (VP) were reduced directly by the polymer to complete its catalytic cycle. Thus clear differences in the roles of redox mediators were found in the oxidation of RNase between LiP and MnP2 (VP).
Reduction of MnP2 (VP) compound I by RNase
Inhibition of VA and Mn2+ oxidation by polymeric substrates
RNase was directly oxidized by MnP2 (VP). The Lineweaver–Burk plots of 1/v versus 1/[VA] at fixed concentrations of RNase, were intercepted on the y-axis (Figure 6), indicating a competitive inhibition pattern. This supports that VA is oxidized at the same binding site as that of the polymeric substrate, RNase. Oxidation of Mn2+ by MnP2 (VP) was inhibited by Poly R-478 non-competitively (results not shown).
Inhibitory effects of RNase on VA oxidation by MnP2 (VP)
Catalytic properties of MnP2 (VP) chemically modified with NBS
Chemically modified MnP2 (VP) was prepared using NBS. The modified MnP2 (VP) exhibited the same Soret and visible absorptions as the unmodified native enzyme (results not shown). On treatment of MnP2 (VP) with NBS, the VA and Poly R-478 oxidation activities were reduced depending on the NBS concentration (Figure 7). However, the activity for Mn2+ oxidation was retained.
Effects of chemical modification of MnP2 (VP) with NBS on oxidation of VA, Mn2+ and Poly R-478
Molecular modelling of MnP isoenzymes from P. ostreatus
VPs share many structural and functional features with LiP and ‘classical’ MnP, which requires Mn2+ as an obligatory reductant for compound II. Figure 8 shows schematic representations of LiP-H8 from Phanerochaete chrysosporium, MnP2 (VP) and MnP3 from P. ostreatus. LiP-H8 and MnP2 (VP) include 12 predominant α-helices. In contrast, MnP3 lacks a B′ helix as in ‘classical’ MnP (H4) from Ph. chrysosporium.
Schematic molecular representation of peroxidase from LiP-H8 from Ph. chrysosporium (A), and MnP2 (VP) (B) and MnP3 (C) from P. ostreatus after homology modelling by ESyPred3D
Reaction mechanism of VPs is characterized by the oxidation of Mn2+ to Mn3+ at the Mn2+-binding site near the haem (Glu-36, Glu-40, Asp-181 for P. ostreatus MnP2) and oxidation of non-phenolic aromatic compounds at a redox active site (Trp-170 for MnP2) exposed on the enzyme surface. However, the detailed mechanisms for the oxidation of polymeric substances by VPs remain to be reported.
We demonstrated that the oxidized form of P. ostreatus MnP2 (VP) was reduced back to the resting state by Poly R-478, with concomitant depolymerization of the dye (Figures 1 and 2). This reaction occurred, regardless of the presence of Mn2+ and a redox mediator VA. Poly R-478 inhibited Mn2+ oxidation by MnP2 (VP) in a non-competitive manner (results not shown). These results are consistent with the mechanism that the oxidation of Mn2+ is catalysed at an Mn2+-binding site, whereas the oxidation of polymers occurs at the enzyme surface. Similar reactivity of MnP2 (VP) for polymeric substrates was also found for RNase (Figures 3–5).
Blocking of the exposed tryptophan residue by NBS was performed to obtain further evidence of a long-range electron transfer pathway in MnP2 (VP). The chemical modification inhibited the oxidation of Poly R-478 and VA by MnP2 (VP), but the Mn2+-oxidizing activity was retained. NBS oxidized tyrosine, histidine, cysteine and methionine, but molecular modelling of MnP2 (VP) demonstrated that the possible amino acid residues involved in substrate binding were Trp-170 and His-82. His-82 is located at the edge of the haem access channel and the binding of bulky substrates to this site is difficult. In addition, site-directed mutagenesis of His-82 in LiP demonstrated that His-82 was not involved in the oxidation of VA . His-239 is the other hypothetical substrate-binding site but, unlike LiP, the multifunctional enzyme, MnP2 (VP), from P. ostreatus has no histidine residue corresponding to His-239, ruling out the involvement of this pathway. Therefore the chemical modification by NBS strongly suggests that NBS blocked the exposed redox active site Trp-170. A steady state kinetic study demonstrated that RNase competitively inhibited the oxidation of VA (Figure 7). These results suggest that the oxidation of VA, Poly R-478 and RNase was catalysed by long-range electron transfer from Trp-170 exposed at the surface of the enzyme.
All the results described so far illustrate that MnP2 (VP) is unique in its catalytic function to oxidize polymeric substrates without redox mediators. The reactivity can be differentiated from that of LiP from Ph. chrysosporium. LiP oxidized Poly R-478 , RNase  and ferric cytochrome c . The oxidation was dependent on the presence of VA. The difference in the dependence on VA for the oxidation of polymers with high redox potential is explained by the acidic microenvironment near the surface of a tryptophan residue. The acidic microenvironment stabilizes the VA–enzyme cation radical complex. Lip-H8 has four acidic amino acids surrounding Trp-171, Asp-165, Glu-168, Glu-250 and Asp-264, whereas P. ostreatus MnP2 (VP) possesses two acidic amino acid residues near Trp-170 (Glu-167 and Glu-249) (Figure 9). The anionic charge near the exposed tryptophan stabilizes the enzyme–VA cation radical complex. The enzyme–VA cation radical complex allows the shift of an unpaired electron from the catalytic centre to the exposed redox active site, accelerating the electron transfer from substrates to the superficial tryptophan residue. Trp-170 in MnP2 (VP) would have enough redox potential to allow the electron transfer from substrates without the binding with VA. The second reason is explained by steric hindrance. Formation of the enzyme–VA cation radical complex is advantageous for a contact with polymeric substrates, since the redox active site can be elongated to the outside by the coupling by VA. LiP would need the binding of VA to avoid steric hindrance with polymeric substrates. A crystal model of LiP-H8 and molecular modelling of MnP2 (VP) showed that Phe-267 near Trp-171 in LiP-H8 protrudes further from the surface compared with Phe-264 in MnP2 (VP) (results not shown). This effect is not observed in reactions with polymers having lower redox potentials like ferrous cytochrome c, which permits the oxidation at the multiple oxidation sites other than the tryptophan residue. The third reason is ascribed to electrostatic repulsion between LiP and substrates caused by anionic charge of four acidic amino acids surrounding Trp-171. The anionic charge around the tryptophan residue in MnP2 (VP) is much smaller than that of LiP-H8. The other possible reason is that LiP requires VA to avoid inactivation by excess H2O2  and radicals. The mechanisms of electron transfer from the exposed tryptophan and roles of VA in polymer oxidation by VPs and LiP await further study.