Description of a Versatile Peroxidase Involved in the Natural Degradation of Lignin That Has Both Manganese Peroxidase and Lignin Peroxidase Substrate Interaction Sites*

Susana Camarero, Sovan SarkarDagger , Francisco Javier Ruiz-Dueñas, María Jesús Martínez, and Ángel T. Martínez§

From the Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Velázquez 144, E-28006 Madrid, Spain

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The biodegradation of lignin represents a key step for carbon recycling on earth because forest ecosystems contain about 150,000 million tons of wood (1). Although the main wood constituent, cellulose, can be utilized by a variety of organisms, its hydrolysis in situ by most of them is hampered by the recalcitrant lignin polymer, which is formed by polymerization of plant p-hydroxycinnamyl alcohols (2). However, the so-called white rot fungi, a group of species from class Basidiomycetes, have developed a remarkable capability for oxidative depolymerization and subsequent mineralization of lignin (3) enabling cellulose utilization by other organisms.

During recent years many studies on lignin biodegradation have been carried out in the fungus Phanerochaete chrysosporium (order Stereales). The first evidence of peroxidase involvement came from inhibition of lignin degradation by catalase (4). Then two peroxidases involved in lignin degradation, the so-called lignin peroxidase (LiP)1 and manganese peroxidase or manganese-dependent peroxidase (MnP), were described (5-7). LiP is characterized by high redox potential enabling oxidation of hardly biodegradable nonphenolic aromatic compounds, such as veratryl alcohol and methoxylated benzenes. P. chrysosporium MnP strictly requires Mn2+ to complete the catalytic cycle, and the chelates of Mn3+ formed can act as efficient oxidizers of phenols and other compounds. Because most units in lignin are nonphenolic (2), LiP was considered as the main enzyme responsible for lignin depolymerization (3). This is still generally accepted, although some evidence suggests that lignin biodegradation could proceed by endwise attack at the phenolic units (8) and that, in addition to Mn3+ chelates, strong chemical oxidants can be generated by MnP (9).

Since 1983-1984 a large amount of information on peroxidases from P. chrysosporium has been accumulated (10-13), and crystal models have been described (14-16). At the time of the LiP model, only the cytochrome c peroxidase crystal structure was available. The information on their molecular structure not only clarified some peculiarities of these enzymes but also contributed to better understanding of some aspects of peroxidase structure and function (17). Despite the above progresses, the mechanism by which lignin is biodegraded is still to be fully understood, and only a limited number of biotechnological applications have been developed. This is related to the fact that the above studies have been focused on a single organism and that little is known about lignin degradation by fungi in their natural environment. Studies on lignocellulose degradation under solid state fermentation (SSF) conditions, which are close to those of natural habitat of white rot fungi, should provide information to contrast that obtained using liquid media (18-20).

The fungus Pleurotus eryngii (order Poriales) has been reported as having the capability to remove lignin selectively when growing on natural substrates and is therefore considered a model organism for studies on biodegradation of lignin in annual plants and related biotechnological applications (21, 22). Peroxidases oxidizing Mn2+ have been reported in liquid cultures of P. eryngii (23), together with the AAO responsible for H2O2 generation in Pleurotus species and laccases (21, 24). However, no typical LiP has been described in this or other Pleurotus species. The aim of the present study was to identify and characterize the peroxidases involved in natural degradation of lignin by this fungus and to investigate the eventual production of LiP. P. eryngii was therefore grown on a lignocellulosic substrate under SSF conditions, and several peroxidases were isolated. Their catalytic properties were investigated using different substrates, and a three-dimensional model for a P. eryngii peroxidase was built using existing crystal models from other fungal peroxidases as templates.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Organisms and Culture Conditions-- P. eryngii ATCC 90787 (IJFM A169), Trametes versicolor IJFM A136, and P. chrysosporium ATCC 24725 (VKM F-1767) were grown on sterile wheat straw under SSF conditions. These are characterized by the presence of enough liquid phase to saturate the solid lignocellulosic substrate, maintaining an air phase between particles. The fungi were grown in an horizontal rotary fermentor, which included six 2-liter bottles containing 125 g of straw (length, 1-2 cm) and 375 ml of water (including inoculum). The inoculation and growth conditions were already described (22). Samples were taken during 2 months to analyze enzymatic activities and substrate degradation. Lignin content was estimated by the Klason method after Saeman's acid hydrolysis and polysaccharide composition by gas chromatography of the acid hydrolyzate, following T-222 and T249 rules (25).

Chemicals-- Reactive Black 5 (C26H21N5O19S6Na4) was obtained from DyStar (Frankfurt, Germany), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) was from Boehringer, and other chemicals were from Sigma-Aldrich.

Enzymatic Activities-- LiP activity was assayed by oxidation of 2 mM veratryl alcohol to veratraldehyde (epsilon 310 9.3 mM-1 cm-1) in 0.1 M tartrate buffer, pH 3, with 0.4 mM H2O2 (controls without H2O2 were included). Mn2+-oxidizing peroxidase was estimated by formation of Mn3+ tartrate (epsilon 238 6.5 mM-1 cm-1) from 0.1 mM MnSO4 in 0.1 M sodium tartrate, pH 5, with 0.1 mM H2O2. Peroxidase activity on phenols was quantified using 2.5 mM syringol (2,6-dimethoxyphenol) (epsilon 469 27.5 mM-1 cm-1 referred to substrate), in 0.1 M sodium tartrate, pH 3, with 0.1 mM H2O2 (23). AAO was determined as the veratraldehyde formed from 5 mM veratryl alcohol in 0.1 M phosphate buffer, pH 6. Laccase was measured with 10 mM syringol in 0.1 M sodium tartrate, pH 5. One activity unit was defined as the amount of enzyme transforming 1 µmol of substrate/min.

Enzyme Purification-- Peroxidases were purified from 15-day-old SSF cultures. Water extracts from the straw treated with the fungus were obtained (by adding 3 liters/bottle and shaking for 1 h at 200 rpm), filtered (0.8 µm), concentrated by ultrafiltration (5-kDa cut-off), and dialyzed against 10 mM sodium tartrate, pH 4.5. The concentrate (approximately 140 ml) was loaded onto a Bio-Rad Q-cartridge (1 ml/min), and retained fractions were eluted with 1 M NaCl. Fractions with peroxidase activity were concentrated, and 1-ml samples applied onto a Sephacryl S-200 HR column (0.8 ml/min). The peroxidase fractions were pooled, concentrated, and dialyzed against 10 mM sodium tartrate, pH 5, and 1-ml samples were applied to a Mono-Q column. Proteins with peroxidases activity were separated using a 0-0.25 M NaCl gradient (30 min, 0.8 ml/min).

Enzyme Characterization-- Protein concentration was determined with the Bradford reagent. Isoelectric focusing was performed in 5% polyacrylamide gels with a thickness of 1 mm and a pH range from 2.5 to 5.5. SDS-polyacrylamide gel electrophoresis was carried out in 12% polyacrylamide gels. Gels were stained with Coomassie Blue R-250. Proteins were deglycosylated using Endo-H from Boehringer. The N-terminal sequences were obtained by automated Edman degradation of 5 µg of protein in an Applied Biosystems 494 pulsed liquid protein sequencer. The steady state kinetic constants were obtained from Lineweaver-Burk plots, and the mean values are presented. The kinetic constants for Mn2+ peroxidase activity were calculated by the formation of Mn3+ tartrate at pH 5. Manganese-independent activities at pH 3 on ABTS (cation radical epsilon 436 29.3 mM-1 cm-1), Reactive Black 5 (epsilon 558 50 mM-1 cm-1), guaiacol (o-methoxyphenol) (oxidation product epsilon 456 12.1 mM-1 cm-1 referred to substrate concentration), syringol, methoxyhydroquinone (methoxybenzoquinone epsilon 360 1.25 mM-1 cm-1), veratryl alcohol, and p-dimethoxybenzene (benzoquinone epsilon 254 21 mM-1 cm-1) were estimated in the presence of EDTA, and kinetic constants were calculated. The Km for the enzyme-oxidizing substrate H2O2 was also obtained at pH 5 (by measuring Mn2+ oxidation) and pH 3 (syringol oxidation). Reciprocal inhibition between Mn2+ and Reactive Black 5 was investigated by following spectrophotometrically the effect of different concentrations of both compounds as inhibitors on the decolorization by peroxidase PS1 of 0.8-25 µM Reactive Black 5 (high concentration caused substrate inhibition) in 0.1 M sodium tartrate, pH 4, and the oxidation of 10-400 µM Mn2+ in 0.1 M sodium tartrate, pH 5 (all reactions contained 0.1 mM H2O2).

HPLC Analysis of p-Dimethoxybenzene Oxidation-- The oxidation of p-dimethoxybenzene (1 mM) was followed spectrophotometrically, using 0.1 M sodium tartrate, pH 3, and 0.2 mM H2O2. Samples (20 µl) from the reaction mixture were analyzed by HPLC using a C18 column (Spherisorb S5ODS2) at 30 °C. A methanol/phosphoric acid (10 mM) gradient (consisting of 10% methanol for 10 min, 10-100% methanol in 6 min, 100% methanol for 4 min, 100-10% methanol in 0.5 min, and 10% methanol for 6.5 min) was used (1 ml/min). Dual wavelength and diode array detectors were used. Standard calibration curves were used for quantitation.

Protein Modeling-- The atomic coordinates of crystal models of LiP-H8 (PDB entries 1LGA and 1LLP), LiP-H2 (1QPA), and MnP1 (1MNP) from P. chrysosporium and ARP-CIP (1ARP) were obtained from the Brookhaven Protein Data Base. The latter peroxidase has been crystallized from both Coprinus cinereus (CIP) (26) and Arthromyces ramosus (ARP) (27). Because the latter is an invalid species (nomen nudum), which could correspond to a Coprinus conidial state, and both proteins share 99% identity, the name ARP-CIP is used here for both of them. The gene coding for protein PS1 of P. eryngii has been cloned, and a preliminary sequence has been obtained (28). From the predicted amino acid sequence a three-dimensional model for the mature protein without Mn2+ was obtained by sequence homology using the program ProMod and refined by CHARMm (the C-terminal region was not modeled) (29). It was based on alignment of sequence of peroxidase PS1 with the above four fungal peroxidases for which crystal models are available. Secondary structure was determined with the DSSP program (30). The above models for P. chrysosporium MnP and LiP were also used for comparison with P. eryngii peroxidase PS1 using programs Swiss-Pdb Viewer and RasMol. Multiple sequence alignment was prepared with PILEUP from GCG package using W2H interface.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

P. eryngii degrades wheat lignin maintaining high cellulose content. This resulted in the increased polysaccharide/lignin ratio shown in Table I. Preferential degradation of lignin is a singular characteristic because most ligninolytic basidiomycetes (including the P. chrysosporium and T. versicolor strains shown in Table I) cause stronger degradation of cellulose than lignin. When the presence of ligninolytic enzymes was investigated in the partially degraded straw no LiP was detected in any case, but AAO, laccase, and Mn2+-oxidizing peroxidase were all produced by P. eryngii and T. versicolor. The high Mn2+-oxidizing activity produced by P. eryngii suggested that the enzyme responsible for this activity could be involved in lignin degradation.

                              
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Table I
Extracellular enzymatic activities and fungal modification of lignocellulose composition
No peroxidase activity on veratryl alcohol (LiP) was detected, and maximal laccase, AAO, and Mn2+-oxidizing peroxidase (MnP) activities are shown as units/100 g initial straw.

The process of peroxidase purification included ultrafiltration of the extract from SSF culture of P. eryngii, followed by Q-cartridge (removing colored compounds from lignin degradation), molecular size exclusion, and ion exchange chromatography. As shown in Fig. 1A, the last purification step resulted in the isolation of two major protein peaks (labeled PS1 and PS3) and a minor one (protein PS2). Both major peaks were electrophoretically homogeneous (Fig. 1B) and were fully characterized. The three proteins exhibited high absorbance at 410 nm and were able to oxidize Mn2+ in the presence of H2O2. The optimum for this reaction was at pH 5. Proteins PS1, PS2, and PS3 differed in Mono-Q retention volume, A410/A280 ratio (4.9, 4.9, and 5.5), isoelectric point (3.67, 3.65, and 3.80, respectively), molecular mass (45, 45, and 42 kDa, respectively), and N termini (VTCATGQTT for PS1 and PS2 and VTCADGNTV for PS3). In the case of protein PS1 a total of 25 amino acid residues were identified, and no double sequences were observed confirming protein purity. Proteins PS1 and PS3 also differed in the N-glycosylation degree (4 and 2%, respectively). A different Mn2+-oxidizing peroxidase produced by P. eryngii when grown in liquid media (23) has a molecular mass intermediate between those of the two major peroxidases isolated here (Fig. 1B) and also differs in the N-terminal sequence.


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Fig. 1.   Purification of P. eryngii extracellular peroxidases from wheat-straw partially delignified by the fungus. A, ion exchange chromatography. In the last purification step (Mono-Q chromatography at pH 5) two major and one minor proteins (PS1 to PS3) with high absorbance at 410 nm and peroxidase activity on different substrates were isolated from straw treated with the fungus under SSF conditions (410- and 280-nm profiles and NaCl gradient are shown). B, SDS-polyacrylamide gel electrophoresis. Peroxidases PS1 and PS3 are shown before (lane 4) and after Mono-Q separation (lane 5, PS1; lane 6, PS3) compared with peroxidase from liquid culture (lane 3) (23) (Coomassie Blue R-250 staining). Lanes 1 and 2 correspond to high and low molecular mass standards.

In addition to their activity on Mn2+, the peroxidases isolated from lignocellulose cultures were able to oxidize substituted phenols, such as guaiacol, syringol, and methoxyhydroquinone. The proteins PS1 and PS2 were able to also oxidize nonphenolic aromatic molecules such as the LiP substrate veratryl alcohol. The fact that peroxidase activity on veratryl alcohol (LiP-type activity) was not detected in the cultures (Table I) is probably due to its inhibition by phenols (or other aromatic compounds in extracts) as described for LiP (19;31). Because veratryl alcohol is a substrate of Pleurotus AAO (and can be oxidized by other fungal enzymes in the presence of mediators), p-dimethoxybenzene was assayed as a more specific LiP substrate. The results of incubation with peroxidases PS1 and PS3, only the first enzyme oxidizing the substrate, are shown in Fig. 2 (A and B). The HPLC analysis revealed benzoquinone as the reaction product by peroxidase PS1 (Fig. 2C). The higher redox potential of peroxidase PS1 was evidenced also by oxidation of some synthetic dyes. Whereas both enzymes oxidized ABTS (PS1 with 3-fold higher specific activity than PS3), only peroxidase PS1 was able to decolorize the Reactive Black 5, a high redox potential azo dye. The optimum for all the manganese-independent reactions was around pH 3. 


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Fig. 2.   Assay of p-dimethoxybenzene oxidation by P. eryngii peroxidases PS1 and PS3. A, peroxidase PS1. Substrate oxidation was evidenced by the increase in absorbance at 254 nm during incubation of 1 mM p-dimethoxybenzene with peroxidase PS1 (the reaction was in 0.1 M sodium tartrate, pH 3, using 0.2 mM H2O2; scans were recorded every 2 min; the molar absorbances of benzoquinone and p-dimethoxybenzene are epsilon 254 21 mM-1 cm-1, and epsilon 286 0.98 mM-1 cm-1, respectively). B, peroxidase PS3. The UV spectrum did not change under the same conditions described in A revealing that p-dimethoxybenzene is not oxidized by this enzyme. C, HPLC analysis (PS1). Benzoquinone formation after p-dimethoxybenzene incubation (20 min) with peroxidase PS1 was confirmed by HPLC using dual wavelength (254 and 286 nm) and diode array detectors, which enabled identification of the quinone formed (the benzoquinone UV spectrum is characterized by maximum at 254 nm) and quantitation of reaction substrate and product.

The steady state kinetic constants of the two major peroxidases for oxidation of Mn2+ to Mn3+ and for manganese-independent oxidation of two phenolic and two nonphenolic aromatic substrates, and the dye Reactive Black 5 are shown in Table II. As already mentioned, the three latter substrates were oxidized only by peroxidase PS1, but both peroxidases also differed in the Km values for the other substrates. These revealed higher affinities for Mn2+ and phenols of PS1 than PS3. Moreover, peroxidase PS1 could oxidize methoxyhydroquinone concentrations below 1 µM, whereas peroxidase PS3 did not exhibit activity below 100 µM substrate. The Km obtained for peroxidase PS1 (17 µM) is lower than found for other enzyme-reducing aromatic substrates (with the only exception of synthetic dyes), revealing substituted hydroquinones as the best natural substrates of P. eryngii peroxidase PS1 from the point of view of enzyme affinity. However, because of the higher turnover number for Mn2+ oxidation, both peroxidases showed the highest efficiency (t/Km) for Mn2+ oxidation (even considering that oxidation of aromatic compounds should involve two one-electron oxidations). The apparent Km values for H2O2, obtained during both Mn2+ oxidation at pH 5 and oxidation of aromatic substrates at pH 3, were low. This corresponds to high affinity of peroxidases for H2O2, but the value obtained at pH 3 could be considered as extremely low compared with other fungal peroxidases. Mutual inhibition between peroxidase PS1 oxidation of the substrates Reactive Black 5 (an azo dye that is not oxidized by Mn3+) and Mn2+ was found. However, the azo dye appeared as a stronger inhibitor because the inhibitor/substrate molar ratio, to cause 40% inhibition, was 125 in the case of Mn2+ inhibition of dye oxidation, and only 0.13 in the case of Reactive Black 5 inhibition of Mn2+ oxidation. The type of inhibition caused by Reactive Black 5 was investigated using different dye concentrations, and although a decrease of the velocity of Mn2+ oxidation was found (the turnover number decreasing from 71 to 43 s-1 in the presence of 32 µM dye), the Km value was maintained (around 50 µM). This suggests two different substrate interaction sites with different affinities that are not affected by the presence of the alternative substrate acting as inhibitor. The inhibitory effect would be caused by the reduction of the oxidized enzyme forms (compounds I and II) during oxidation of the substrate acting as inhibitor. Finally, it is possible to mention that the stronger inhibitory effect caused by Reactive Black 5 agrees with the higher peroxidase affinity for this substrate, compared with the affinity for Mn2+.

                              
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Table II
Steady state kinetic constants of the two fungal peroxidases (PS1 and PS3) purified from straw partially delignified by P. eryngii (mean values)

Because the above results showed that peroxidase PS1 shared LiP and MnP-type catalytic properties, as well as some characteristics of other peroxidases oxidizing phenolic substrates, a three-dimensional model was built for the mature protein (this model includes the two helical domains characteristic of all peroxidases). The four fungal peroxidases for which crystal structures are available were used as templates for homology modeling, and a multiple alignment of their corresponding sequences together with the PS1 sequence that was modeled is shown in Fig. 3. These peroxidases are P. chrysosporium MnP1 (58% sequence identity with PS1) and isoenzymes H8 (60%) and H2 (62%) of LiP, and Coprinus ARP-CIP (52%). The model obtained showed good geometry with root mean square deviations in bond angles and distances of 2.31 ° and 0.013 Å, respectively. It includes 12 predominantly alpha  helices, the position and size of which is indicated in Fig. 3. The helix B"corresponds to helix B' of LiP (14), with both being longer than helix B' of MnP1. A short helix at the position of helix B' of P. eryngii peroxidase PS1 was described in ARP-CIP (27), and some helical conformation at this position can be observed also in LiP. Therefore, the three latter peroxidases could include 12 helices, compared with only 11 helices of P. chrysosporium MnP1. The peroxidase PS1 conserves some structurally important elements including disulphide bridges (the last one is not included in the model because it links the C-terminal region to helix I) and putative Ca2+ sites (distal Ca2+ linked by oxygens of Asp48, Gly66, Asp68, and Ser70; and proximal one by those of Thr176, Asp193, Thr195, Thr198, and Asp200), characteristic of all peroxidases in classes II and III (17). The peroxidase PS1 model shows the highest coincidence in protein folding and helical topology and the lowest root mean square distance between backbone Calpha with the LiP-H8 and ARP-CIP models. However, when superimposed with P. chrysosporium MnP1 differences were found in two superficial loops (PS1 Thr57-Ala59 and MnP1 Leu228-Thr234).


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Fig. 3.   Multiple alignment of the amino acid sequence from P. eryngii peroxidase PS1 (mature protein) corresponding to the three-dimensional model built (details in Figs. 4 and 6), together with sequences from P. chrysosporium LiP (isoenzymes H8 and H2) and MnP1, and Coprinus ARP-CIP used for homology modeling. The atomic coordinates of the four latter proteins were used as templates to obtain the three-dimensional model (Protein Data Bank entry 1BQW) for the P. eryngii peroxidase PS1 including two helical domains, which contain all the catalytically important residues (the C-terminal region, which has variable length in different peroxidases, was not modeled). The alignment was built by PILEUP from GCG package, and the amino acid residues identical to those found in the PS1 sequence are depicted in white on black (the distal and proximal histidine residues are marked with an asterisk). The position of the 12 helices in the PS1 peroxidase predicted by the DSSP program is indicated on the corresponding sequence. The mature ARP-CIP includes an extra N-terminal sequence of 8 amino acids (QGPGGGGS), which in other fungal peroxidases corresponds to the signal peptide.

Some aspects of the model directly related to catalytic activity were considered more in depth. The amino acid residues at the heme pocket of P. eryngii peroxidase PS1 were the same found in other fungal peroxidases (with the only exception of ARP-CIP): Arg43, Phe46, His47, Glu78, and Asn84 in the distal side and His175, Phe192 (Leu201 in ARP-CIP), and Asp237 in the proximal side. Fig. 4 (left side) shows the opening of the main heme access channel in P. eryngii peroxidase PS1 (top), P. chrysosporium LiP-H8 (center), and MnP1 (bottom). This channel enables the access of hydroperoxides to the distal side of heme. Moreover, it has been postulated that some of the edge residues are involved in oxidation of aromatic substrates by LiP and horseradish peroxidase. As shown in Fig. 4, the P. eryngii peroxidase PS1 conserves six of the eight residues delimiting the channel edge in LiP-H8 (and one of the two differences is the substitution of Ile85 by Leu85). However, the heme channel of P. chrysosporium MnP1 differs from that of LiP-H8 in five residues. Fig. 4 (right side) also shows the narrow Mn2+ channel of P. eryngii peroxidase PS1 and P. chrysosporium MnP1 directly on the internal heme propionate, where the cation is fixed by the three acidic amino acid residues (one aspartate and two glutamates) delimiting the channel edge. Finally, it is interesting that Trp170 of peroxidase PS1 occupies a position at the protein surface similar to that of LiP Trp171 (that could transfer electrons to heme because of topological proximity), whereas a similar residue is absent from the ARP-CIP and MnP1 models.


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Fig. 4.   Amino acid residues at the opening of the main heme-channel of P. chrysosporium LiP-H8 reported to be involved in aromatic substrate (AS) oxidation (center) and residues forming the small Mn2+ channel of P. chrysosporium MnP1 (bottom) compared with residues occupying the same positions in P. eryngii peroxidase PS1 (top) (as van der Waals' spheres). The three acidic residues responsible for Mn2+ binding are indicated in yellow. His82 and Gln221/222, reported as involved in veratryl alcohol binding to LiP (His82 also in electron transfer to heme; see Fig. 6), and Phe148, occupying a position similar to that of horseradish peroxidase Phe142 involved in aromatic substrate interaction, are indicated in orange. The atomic coordinates correspond to Brookhaven Protein Data Base entries 1BQW (three-dimensional model for peroxidase PS1 built from sequence in Fig. 3, modeled without Mn2+), 1LLP, and 1MNP (LiP-H8 and MnP1 crystal models).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

LiP and MnP of P. chrysosporium have been considered as two models for all ligninolytic peroxidases. However, this study shows that one of the two major peroxidases produced by P. eryngii during lignin degradation under natural growth conditions can be considered as representative for a third type of ligninolytic peroxidase, different from other microbial, plant, or animal peroxidases (17). As shown in Fig. 5, this versatile peroxidase is able to perform both the oxidative reactions characteristic of P. chrysosporium LiP, i.e. the oxidation of nonphenolic aromatic substrates via aromatic radicals, and MnP, i.e. the oxidation of Mn2+ to Mn3+. Its affinity for H2O2 and Mn2+ was higher than reported for P. chrysosporium peroxidases, but the turnover number for Mn2+ and the affinity and turnover number for veratryl alcohol were lower (32-34). Moreover, the P. eryngii peroxidase efficiently oxidizes substituted phenols (the affinity for methoxyhydroquinones being even higher than for Mn2+), which cannot be oxidized by the P. chrysosporium peroxidases. This is because these compounds inactivate LiP in the absence of veratryl alcohol (31), whereas compound II of P. chrysosporium MnP needs to be reduced by Mn2+ to close the catalytic cycle (phenols are indirectly oxidized by Mn3+) (35). In addition to the above substrates, the P. eryngii peroxidase PS1 can oxidize two compounds, alpha -keto-gamma -methylthiobutyric acid and p-dimethoxybenzene, which were those used for LiP detection (7) and for demonstration of oxidation mechanism by this enzyme (36). The oxidation of alpha -keto-gamma -methylthiobutyric acid, a substrate characteristic of strong one-electron abstracting agents (such as LiP and hydroxyl radical), to ethylene by peroxidase PS1 has been reported very recently (37). The results of p-dimethoxybenzene oxidation to benzoquinone confirmed that the P. eryngii peroxidase has high redox potential and acts on aromatic substrates by the same mechanism described for LiP, i.e. via an aromatic cation radical formed by one electron oxidation of the benzenic ring (36). Finally, Reactive Black 5 is oxidized by the P. eryngii peroxidase, the reaction being inhibited by high Mn2+ concentrations. This azo dye cannot be oxidized by P. chrysosporium MnP because its redox potential is higher than that of Mn3+ tartrate nor by LiP in the absence of veratryl alcohol because of rapid inactivation (38). However, no inactivation is produced in the case of peroxidase PS1. The Reactive Black 5 also acts as a very efficient noncompetitive inhibitor of Mn2+ oxidation by the P. eryngii peroxidase. This result agrees with the noncompetitive inhibition by Mn2+ of the oxidation of dyes and veratryl alcohol by two similar peroxidases recently isolated from liquid cultures of Pleurotus and Bjerkandera species (38-40) and supports the existence in these proteins of a specific substrate interaction site for Mn2+, different from that involved in oxidation of other peroxidase substrates.


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Fig. 5.   Scheme of catalytic cycles of P. chrysosporium LiP (A) and MnP (B) compared with Pleurotus peroxidase (C) (panels A and B were adapted from Ref. 13). Cycles include two-electron oxidation of enzyme by hydroperoxides (ROOH) to compound I, a complex of high valent oxo-iron and porphyrin cation radical ([Fe4+ = O P·+]), followed by two one-electron reductions of compound I to compound II ([Fe4+ = O P]) and native enzyme ([Fe3+ P]), producing two oxidations of the electron donor. They differ in electron donors, which can be: (i) aromatic compounds (AH) for LiP (generally nonphenolic because the enzyme is inactivated during oxidation of phenols); (ii) Mn2+ for MnP (although phenols can reduce compound I and are indirectly oxidized by Mn3+); and (iii) Mn2+ and phenolic or nonphenolic aromatic compounds for the Pleurotus peroxidase. The optimum for Mn2+ oxidation in all above reactions was around pH 5, and that for direct oxidation of aromatic substrate was around pH 3.

No crystal structures for P. chrysosporium LiP have been obtained containing veratryl alcohol, which would provide invaluable information for identification of substrate interaction site. However, when the enzyme was crystallized (14, 15),it was suggested that oxidation of veratryl alcohol could be produced at the heme access channel, and some amino acid residues were postulated to be involved in substrate binding and electron transfer. Poulos et al. (14) modeled the veratryl alcohol molecule at the heme edge of LiP-H8, H-bonded to His82 and Gln222. These two residues are conserved in the P. eryngii peroxidase PS1, which has a remarkably similar heme access channel as shown in the three-dimensional model obtained (Fig. 4, top and center). The presence of different amino acid residues at the above two positions (Fig. 4, bottom) probably prevents direct oxidation of aromatic substrates by MnP, together with other differences resulting in a more polar channel environment. It is interesting that Phe142 of horseradish peroxidase, which occupies a position similar to that of Phe148 of P. eryngii peroxidase and LiP-H8, has been reported to be involved in aromatic substrate interaction (41, 42). Despite the possibility of direct electron transfer from veratryl alcohol at the above binding site to heme as was postulated in P. chrysosporium LiP, substrate oxidation via long range electron transfer (LRET) should also be considered taking into account the distance between channel opening and heme edge. This was suggested by Schoemaker et al. (43), who proposed one of the LRET pathway shown in Fig. 6 (bottom). This initiates in LiP His82, mentioned as involved in veratryl alcohol binding, and proceeds via Pro83 and Asn84, the latter being H-bonded to the distal histidine (His47). Such a LRET pathway does not exist in P. chrysosporium MnP, but the three-dimensional model built showed that the His82 of P. eryngii peroxidase was also exposed at the heme channel opening (Fig. 4, top) and connected by a similar pathway with the distal histidine (PS1 His47) (Fig. 6, top). The difference is that PS1 Ala83 occupies the position of LiP Pro83, resulting in a different position of His82 side chain at the protein surface. Recently Trp171 of P. chrysosporium LiP has been described as being involved in the catalytic cycle of the enzyme via a different LRET pathway (Fig. 6, bottom), which does not exist in P. chrysosporium MnP (44). However, Trp170 of P. eryngii peroxidase PS1 occupies a similar position at the protein surface and it is linked to heme by a LRET pathway as described in LiP (Fig. 6, top). Therefore, this aromatic residue could be involved in oxidation of some substrates by peroxidase PS1, whereas other substrates would be oxidized at the heme channel, as recently shown for P. chrysosporium LiP (45).


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Fig. 6.   Stereo views showing two hypothetical LRET pathways to heme in P. eryngii peroxidase PS1 (top) and P. chrysosporium LiP isoenzyme H8 (bottom) from His82 at heme-channel edge (via distal His) and from PS1 Trp170 or LiP Trp171. The atomic coordinates correspond to Brookhaven Protein Data Base entries 1LLP (LiP-H8 crystal model) and 1BQW (three-dimensional model for peroxidase PS1 built from the sequence in Fig. 3).

The ability to directly oxidize Mn2+ to Mn3+ is a unique characteristic of Mn2+-oxidizing peroxidases produced by P. chrysosporium and many other white rot fungi (46). Other enzymes could oxidize Mn2+ via superoxide anion radical, such as that generated by redox cycling (47). The existence of a Mn2+ binding site in P. chrysosporium MnP was predicted in theoretical models of the enzyme built by sequence homology using LiP as a template (48), as well as in the first crystal structure obtained (16). Then it was indirectly confirmed by site-directed mutagenesis of MnP Glu35, Glu39, and Asp179 (32), and direct evidence obtained by x-ray diffraction of the enzyme crystallized in the presence of Mn2+ (49). As shown in Fig. 4, the P. eryngii peroxidase PS1 has the same three acidic amino acid residues (Glu36, Glu40, and Asp181) forming a small channel directly on the internal heme propionate that acts as first electron acceptor for Mn2+ oxidation by these peroxidases. The absence of the three above residues in P. chrysosporium LiP (Ala36, Glu40, and Asn182 at the corresponding positions) results in inability to bind and oxidize Mn2+ (Fig. 4, center). The pH optimum for oxidation of Mn2+ (pH 5) is higher than for direct oxidation of aromatic substrates (pH 3) by the P. eryngii peroxidase. This is because the three acidic residues and the internal heme propionate at the Mn2+ interaction site should be dissociated to bind the cation. The low pH for manganese-independent oxidations is probably due to increased redox potential of heme at low pH values, but the involvement of an acidic amino acid residue in its protonated form has also been suggested (14).

No typical LiP is produced by Pleurotus species, but several Mn2+-oxidizing peroxidases have been reported. Those from P. pulmonarius cultures on wheat straw (18) and P. ostreatus on sawdust (MnP2) (50) probably correspond to the same peroxidase described here, because they have the same N-terminal sequence and catalytic properties. Peroxidases similar to that produced by P. eryngii in liquid culture (GenBank accession number AF007223 and its allelic variant AF007224) (23, 51) are produced by P. pulmonarius in liquid culture (18) and by P. ostreatus in both liquid (52) and sawdust cultures (MnP1) (50) (N terminus ATCADGRTT). In addition to these peroxidases oxidizing both Mn2+ and aromatic substrates and dyes, the peroxidase PS3 from P. eryngii and a peroxidase from liquid culture of P. ostreatus (53) (differing in N terminus) are closer to P. chrysosporium MnP. Production of specific isoenzymes on natural substrates has also been reported in other fungi (18-20), but the physiological/ecological significance of the different peroxidase isoenzymes remains to be established. Recently a MnP with some manganese-independent activity has been described in Poria (synonym: Ceriporiopsis) subvermispora (54), but it has low sequence homology with P. eryngii peroxidases and is not able to oxidize veratryl alcohol. However, enzymes with catalytic properties similar to Pleurotus versatile peroxidases (23) have been found in Bjerkandera adusta (55) and Bjerkandera sp. (40), the latter being described as a MnP-LiP "hybrid" enzyme. Some aspects of the three-dimensional model shown here suggests that the catalytic properties of this third type of ligninolytic peroxidase are related to a hybrid molecular structure including MnP and LiP-type features. Site-directed mutagenesis studies are necessary to confirm substrate interaction sites and LRET pathways (as proposed from His82 and Trp170) in P. eryngii peroxidases. This will be facilitated by the recent expression in Emericella nidulans of the gene encoding a P. eryngii peroxidase.

    ACKNOWLEDGEMENTS

We thank L. Caramelo (Sigma-Aldrich, Spain) for stimulating discussions about LiP-type peroxidases, F. Guillén for help in quinone analyses, B. Böckle for contribution to kinetic constant determination, A. Heinfling (Technical University of Berlin, Germany) for a sample of Reactive Black 5, and J. A. Field (Wageningen Agricultural University, The Netherlands) for information about Bjerkandera sp. peroxidases. S. S. acknowledges the European Fellowship (Human Capital and Mobility programme) for supporting his stage at Centro de Investigaciones Biológicas. We thank J. Varela for N-terminal sequencing, A. Díaz for DNA sequencing, and A. Guijarro and T. Raposo for technical assistance.

    FOOTNOTES

* This work was supported by European Contract AIR2-CT93-1219 and Project BIO96-393 of the Spanish Biotechnology Programme.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (code 1BQW) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.

Dagger Present address: University of Westminster, Div. of Biotechnology, 115 New Cavendish St., W1M 8JS London, UK.

§ To whom correspondence should be addressed. Tel.: 34915611800; Fax: 34915627518; E-mail: cibm149{at}fresno.csic.es; URL address: http://www.cib.csic.es/~lignina/lignina_en.html.

    ABBREVIATIONS

The abbreviations used are: LiP, lignin peroxidase; AAO, aryl-alcohol oxidase; ABTS, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate); ARP, Arthromyces ramosus peroxidase; CIP, Coprinus cinereus peroxidase; LRET, long range electron transfer; MnP, manganese peroxidase; SSF, solid state fermentation; HPLC, high pressure liquid chromatography.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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