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
Sarkar
,
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 |
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.
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INTRODUCTION |
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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EXPERIMENTAL PROCEDURES |
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
(
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
(
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)
(
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
436 29.3 mM
1
cm
1), Reactive Black 5 (
558 50 mM
1 cm
1), guaiacol
(o-methoxyphenol) (oxidation product
456 12.1 mM
1 cm
1 referred to substrate
concentration), syringol, methoxyhydroquinone (methoxybenzoquinone
360 1.25 mM
1
cm
1), veratryl alcohol, and p-dimethoxybenzene
(benzoquinone
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 |
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.
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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.
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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
254 21 mM 1 cm 1,
and 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.
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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)
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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
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 C
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.
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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).
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 |
DISCUSSION |
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,
-keto-
-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
-keto-
-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.
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.
 |
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