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INTRODUCTION |
White rot fungi degrade the aromatic polymer lignin with
extracellular oxidative enzymes. These enzymes include peroxidases and/or laccases (1, 2). The role of each enzyme in this complicated
process is an active area of research and debate. The most studied
white rot fungus, Phanerochaete chrysosporium, produces two
types of peroxidases, manganese peroxidase
(MnP),1 and lignin peroxidase
(LP) (3, 4), but no detectable laccase. The heme-containing peroxidases
undergo the classical catalytic cycle, where
H2O2 first oxidizes the native (ferric)
enzyme, forming compound I (5). Two reducing substrates then donate two
electrons, one each, to complete the catalytic cycle via the formation
of a compound II intermediate (5).
MnP and LP are similar in structure (6-8) but distinguished and unique
in the nature of their reducing substrates. MnP can oxidize phenolic
substrates, but compound II exhibits specificity for complexed
Mn2+, oxidizing it to Mn3+ (9).
Mn3+, in turn, is able to oxidize a wide range of phenolic
substrates including phenolic lignin (9). In contrast, LP oxidizes a
variety of phenolic and nonphenolic aromatic compounds (10, 11). How these two enzymes interact with their ultimate substrate, lignin, has
been intensely investigated. Several studies have shown that the pore
size of the plant cell wall does not allow for direct contact between
the peroxidases and lignin (12-14). For MnP, trivalent Mn is proposed
to deliver the oxidizing equivalents of the heme active site to the
aromatic substrate (9). For LP, the secondary metabolite, veratryl
(3,4-dimethoxybenzyl) alcohol has been proposed to play a similar role
(15). This alcohol has been shown to facilitate the oxidation of many
compounds in vitro (16-18). This observation led these
investigators to speculate that the veratryl alcohol cation radical is
the low molecular weight mediator in the case of LP. However, the life
span of the veratryl alcohol cation radical is too short to permit
diffusion to distant sites (19). Therefore, LP may be involved in the
oxidation of the soluble, partially degraded lignin fragments. However,
the possibility that LP can directly oxidize lignin in the partially
degraded cell wall cannot be excluded.
The present study maps the site of electron transfer for LP in respect
to substrate size. Two likely substrate-binding sites have been
proposed. One is the so-called heme access channel (8), which allows
for direct interaction between the substrate and the heme. Residues
Ile85, Val184, Gln222,
Phe148, His82, Glu146, and
Asp183 are located in this channel. This channel is
sterically restricted and would not allow access to large bulky
(lignin) substrates. The other site, Trp171, is located at
the enzyme surface and would only allow for long range electron
transfer (20). We have designed a tetrameric nonphenolic lignin model
compound, which does not fit into the heme access channel (Fig.
1), and determined the initial products of its oxidation. The product profile indicates that the site of
electron transfer of LP is very exposed. Site-directed mutagenesis studies where Glu146 and Trp171 were altered
indicate that the site of electron transfer is Trp171.

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Fig. 1.
Structure of LP isozyme H8 and the tetramer
model. The figure shows the van der Waals surface of LP
(blue) and the tetramer (yellow). The
left structure shows Glu164
highlighted in green near the heme access channel
with the heme shown in red. The right structure
shows Trp171 highlighted in green
along with the tetrameric model. Trp171 and
Glu164 are on opposite sides of the protein. The figure was
constructed using WebLab ViewerPro and coordinates of Poulos et
al. (8).
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MATERIALS AND METHODS |
Chemicals--
Ammonium cerium(IV) nitrate and
2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) were purchased from
Aldrich. The oligonucleotides for site-directed mutagenesis were
synthesized by Integrated DNA Technologies, Inc. (Coralville, IA).
H2O2 was purchased from VWR and prepared
fresh daily; the concentration was determined spectrophotometrically at
240 nm using an extinction coefficient of 39.4 M
1 cm
1 (21). The tetrameric
lignin model and its carbonyl degradation products (see Fig. 2) were
synthesized and purified as described in Ref. 22. The tetrameric
compound is a mixture of two diasteromeric compounds (ratio 1:1)
because of the presence of R and S configurations in the terminal
position. All other chemicals were commercially available and used
without further purification.
Enzymatic and Chemical Oxidation of the
Tetramer--
Incubations of the tetramer (50 µM) with
fungal LP isozyme H2 and wild type or mutated recombinant LP H8 (1 µM) were performed in 50 mM sodium tartrate
buffer (pH 3.5). The reaction was initiated by the addition of
H2O2 to yield a final concentration of 0.2 mM. Products were also analyzed over a longer time period.
In these experiments, the reaction mixture volume was 15 ml and
initially contained 110 µM tetramer and 1 µM LP isozyme H2 in 50 mM sodium tartrate
buffer (pH 3.5). The reaction was initiated with the addition of 10 µl of H2O2 yielding a final concentration of
0.05 mM. After initiation of the reaction, an additional
aliquot of enzyme (0.5 nmol) and H2O2 (150 nmol) were added in every 2 min to yield a final concentration of 1.5 µM and 0.14 mM, respectively. Aliquots (1 ml)
were removed at different time points from the reaction mixture and
added to 2 ml of acetonitrile and analyzed by HPLC. When the
concentrations of the products were determined, results were corrected
by the dilution factors of the reaction mixture because of the
continuous addition of enzyme and H2O2.
Oxidation of the tetramer (100 µM) by 10 µM
DDQ was performed in 1 ml of dioxane containing one drop of methanol as
described by Fenn and Kirk (23). Oxidation of 100 µM
tetramer by Ce4+ (15 µM) was carried out in 1 ml of distilled water that was acidified by a few drops of concentrated
sulfuric acid.
Identification of the Oxidation Products of the Tetramer--
As
described above, reaction mixtures were sampled, and the reaction was
quenched by the addition of one volume of acetonitrile. This aliquot
was injected onto a reverse phase Discovery C18 column (15 cm × 4.6 mm) with 5 µm of pore size (Supelco, Bellefonte, PA). Samples
were eluted at 0.8 ml/min with a 0-95% step gradient of acetonitrile
in water over 40 min. Products were monitored at 280 nm and identified
by comparison to retention times of authentic standards and by mass spectrometry.
Liquid chromatography-mass spectrometry was performed using a
Model 1100 series HPLC (Hewlett Packard, Palo Alto, CA) coupled to a
Mariner orthogonal acceleration/time of flight mass spectrometer (Perseptive Biosystems, Framingham, MA) equipped with an atmospheric pressure chemical ionization source. The HPLC system was equipped with
a column with a stationary phase identical to that described above.
Mass spectra were acquired in positive ion mode using a nebulizer
temperature of 350 °C.
Mutagenesis, Heterologous Expression, and Enzyme
Purification--
The mutagenesis (E146S) of the pET21aH8(+) was
described previously by Ambert-Balay et al. (24).
Mutagenesis to obtain W171S was performed by a QuickChange
site-directed mutagenesis kit, and protocol was provided by Stratagene
(La Jolla, Ca). The following oligonucleotides were used where the
mutated sequences are underlined: 5'-CTCGAGCTTGTCTCGATGCTCTCC-3';
5'-GGAGAGCATCGAGACAAGCTCGAG-3'. The mutations were
confirmed by DNA sequencing. The plasmid encoding the mutated LP was
transformed into Escherichia coli strain
BL21(DE3)pLysS. The recombinant wild type and mutant peroxidases were
refolded and purified as described previously (24).
Wild type fungal LP isozymes H2 and H8 were purified as described
previously (25). The concentrations of the peroxidases were determined
spectrophotometrically at 409 nm using extinction coefficients of 169 mM
1 cm
1 and 168 mM
1 cm
1 for LP isozymes H2 and
H8, respectively (26). The purified preparations exhibited an
RZ ratio (A409/A280) of least 3.
Identification of the Tetramer Oxidation Products from Single
Turnover Experiments--
The stopped-flow apparatus was purchased
from KinTek Instruments (State College, PA). All reactions were run at
28 °C. To generate compound I, 30 µM LP isozyme H2 in
150 mM sodium tartrate (pH 3.5) in one syringe was mixed
with 28 µM H2O2 in the second syringe. The reaction mix was aged for 1.5 s. This freshly
prepared compound I was then mixed with the tetrameric model compound, which was in the third syringe at 300 µM. The reaction
mixture (2 ml) was complete within 1 min as monitored by changes in
Soret absorbance. The sample was then collected and analyzed
immediately by HPLC.
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RESULTS |
Identification of Oxidation Products--
Incubation of the
tetramer with fungal LP isozyme H2 and H2O2
resulted in cleavage of the tetramer without the addition of the
putative mediator, veratryl alcohol. Previous work (10) had shown that
the predominant products from LP-catalyzed oxidation of lignin models
are that of C
-C
cleavage. The predicted products from such cleavage are shown in Fig.
2. Incubation under multiple turnover
conditions resulted in formation of four products as detected by HPLC
(Fig. 3A), which were
identified by liquid chromatography-mass spectrometry. The mass spectra
and the corresponding retention times of the oxidation products
were compared with those of the authentic standards (Table
I). The proposed products from C
-C
cleavage are also intermediates in
the synthesis of the tetramer (22) and consequently were available as
known standards. As shown in Table I, three of the four products were identical to the standards and were identified as the trimeric, dimeric, and monomeric aldehydes. The fourth predicted product, labeled
2 in Fig. 3, did not readily ionize and thus was not easily identified
by LS/MS. Previous work had also shown that oxidation of
C
alcohols to ketones was also a predominant
reaction of LP-catalyzed reactions (10). We thus suspected that unknown 2 is a products where one of the C
alcohols was oxidized to the corresponding carbonyls. To test this possibility, we incubated the tetramer (100 µM) with a chemical oxidant specific
for oxidizing C
alcohols to the ketone/aldehyde, DDQ
(23). The oxidation was carried out with 10-fold less DDQ relative to
the tetramer to ensure that only one product was formed. Analysis by
HPLC (Fig. 3B) indicated that the product generated by DDQ
had an identical retention time as that of the unknown degradation
product. This is consistent with unknown 2 as one of the four possible
tetrameric carbonyl compounds.

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Fig. 2.
Structure of the tetrameric lignin model
compound and its degradation products. The arrows drawn
in the structure show location of cleavage. Arrows leading
away from these smaller arrows point to products formed from
cleavage at that site.
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Fig. 3.
The HPLC profile of the tetrameric lignin
model compound degradation by LP isozyme H2 (A) or
with the C oxidizer DDQ
(B). Peaks labeled in figure
correspond as follows: 1, tetrameric model compound;
2, tetrameric carbonyl; 3, trimeric aldehyde;
4, dimeric aldehyde; and 5, monomeric aldehyde
compound. See "Materials and Methods" for HPLC conditions.
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Table I
Mass spectra and HPLC retention times of the various lignin model
compounds
See "Materials and Methods" for HPLC conditions.
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Time Course of Oxidation--
To determine the product/precursor
relationship of the four products, the time course of product formation
from the tetramer was determined. Our initial data (not shown)
indicated that extended incubations where H2O2
was in excess of the tetramer did not result in complete oxidation of
the tetramer. This is consistent with our previous results showing
enzyme inactivation with poor substrates of LP (10). Thus, to extend
the time course of tetramer oxidation, the enzyme and
H2O2 were added to the reaction multiple times in small aliquots every 2 min (see "Materials and Methods"). During the 30-min incubation, all four products were detected at every time
point (Fig. 4A). Because
subsequent experiments were performed with recombinant enzyme produced
by the cDNA-encoding isozyme H8, similar time course
experiments were performed with fungal isozyme H8. The same results
were obtained (Fig. 4B).

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Fig. 4.
Time course of the oxidation of the
tetrameric lignin model compound by LP isozyme H2 (panel
A) and isozyme H8 (panel B). Symbols
are as follows: , tetrameric model compound; , tetrameric
carbonyl; , trimeric aldehyde; , dimeric aldehyde; and ,
monomeric aldehyde. Products were identified by liquid
chromatography-mass spectrometry and quantitated by HPLC using standard
curves generated from synthetic standards.
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The results also suggest that the oxidation products are substrates for
further LP catalysis. We thus determined whether the trimer, dimer, and
monomeric (aldehyde) models were substrates for LP. These models were
incubated under the same conditions as used in the experiment of
tetramer oxidations. The products were again identified and quantified
by HPLC (Table II). The trimeric and
dimeric aldehydes were further oxidized, whereas the monomer was not a
substrate for isozyme LPH2. The oxidation of the trimeric compound
resulted in formation of both dimeric and monomeric products, whereas
the dimer yielded only the monomeric aldehyde.
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Table II
Oxidation of the trimeric, dimeric, and monomeric carbonyls by LP
isozyme H2
LP (1 µM) was incubated with 50 µM tetramer
in 50 mM sodium tartrate buffer, pH 3.5. Reactions were
initiated by addition of H2O2 (0.2 mM
final).
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Effect of Veratryl Alcohol--
Several previous studies reported
the stimulatory effect of veratryl alcohol on the oxidation of a
variety of chemicals with ranging from aromatic monomers to polymeric
lignin. Therefore, we also tested the effect of veratryl alcohol on the
oxidation of this tetrameric lignin model. Fig.
5A shows the decrease of tetramer in the absence and presence of veratryl alcohol over a 4-min
time period. The addition of veratryl alcohol enhanced the rate of
tetramer degradation. Approximately 70% of the tetramer was degraded
in the presence of veratryl alcohol during the first 30 s, whereas
only 50% of the tetramer disappeared without veratryl alcohol in the
same interval. The product profile was similar with or without added
veratryl alcohol. The fate of veratryl alcohol was also followed by
HPLC during this experiment. All of the veratryl alcohol was oxidized
to veratraldehyde within 2 min (Fig. 5B).

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Fig. 5.
Effect of veratryl alcohol on the
oxidation of the tetrameric lignin model. Panel A, decrease
of the tetrameric lignin model in the absence ( ) and presence ( )
of 10 µM veratryl alcohol. Panel B, decrease
in veratryl alcohol ( ) and increase in veratraldehyde ( ).
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Products Formed from Single Turnover--
The time course
experiment shown in Fig. 4 suggested that LP equally oxidized all four
possible cleavage sites. If LP acts only as an "exo"
lignin-degrading enzyme, then the time course would first show the
formation of the trimer, concomitant with formation of the monomer.
This would then be followed by formation of dimers. To further
investigate whether LP could act as an "endo" enzyme yielding also
dimers as the initial product, single turnover experiments were
performed. Single turnover conditions were attained in a three-syringe
stopped-flow apparatus as described previously (27). Slightly less than
one equivalent of H2O2 was preincubated with LP
isozyme H2; this ensured only single turnover. The reaction mixture
then aged for 1.5 s to permit complete formation of compound I. The second push of the stop flow then mixed the freshly prepared compound I with the tetramer. This mixture was collected and
immediately analyzed by HPLC. As observed under steady state
conditions, all four oxidation products were detected (Table
III). The products were roughly similar
in concentration.
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Table III
Comparison of the identified products of the tetrameric model compound
during a single turnover of LP isozyme H2 and with Ce4+
Single turnover experiments were performed in the stopped flow as
described under "Materials and Methods." LP (30 µM)
was first incubated with 28 µM H2O2
resulting in the formation of compound I. This was then mixed with the
tetramer (final concentration of 100 µM).
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Oxidation by Ce4+--
Detection of all four possible
oxidation products in single turnover experiments would indicate that
no specificity existed with LP-catalyzed reactions. To corroborate
these results, we also determined the product formed from oxidation of
the tetramer with a chemical oxidant. As shown in Table III, oxidation
of the tetramer with a substoichiometric amount of Ce4+, a
single electron oxidant, resulted in the formation of all four possible products.
Characterization of Mutants of LP--
Two substrate-binding sites
have been suggested for LP, the so-called heme access channel, and a
more surface-exposed site was suggested for Trp171
(8, 28). We investigated the nature of the substrate-binding site using
the tetrameric model as the substrate. Mutants E146S (heme access
channel) and W171S (surface residue) were generated in the recombinant
mutant LP (isozyme H8). Their activity with the tetramer was then
examined and compared with wild type LP. The product profile from
oxidation of the tetramer by the fungal H8 and the recombinant H8 was
similar to that of fungal isozyme H2. Mutant E146S, located at the
heme-access channel, was also able to oxidize the tetramer. The product
profile was similar to the wild type enzyme (Table
IV). In contrast, oxidation of the
tetramer was completely inhibited by the mutation of W171S (Table IV).
However, as reported by Doyle et al. (28), the oxidation of
ABTS was not inhibited by this mutation (data not shown).
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Table IV
Oxidation of the tetrameric lignin model compound by the wild type and
mutated LP isozyme H8
Incubation conditions were the same as those described in Table II.
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DISCUSSION |
Results from the present study help resolve the issue of whether
LP can degrade polymeric lignin without redox mediators and have also
identified the site of electron transfer for polymeric substrates.
These results have relevance on the role of LP in lignin degradation, a
question still debated after the discovery of LP 17 years ago. This
debate is partly because of the finding that many lignin-degrading
fungi apparently do not contain LP (29) and because of the relatively
slow rate of this enzyme with the substrate lignin (30). Because LP is
proficient in oxidizing low molecular weight models of lignin, some
have proposed that the role of LP in lignin degradation is solely for
the oxidation of smaller lignin fragments (30), generated by MnP
catalysis. The ability of LP to bind to its putative substrate, lignin,
has also been questioned. Research from a number of laboratories has shown that the pores of the wood matrix are not large enough to allow
access to enzyme (12-14). However, similar to how MnP can degrade
lignin through utilizing Mn2+/Mn3+ as a
mediator, Harvey et al. (15) suggest that veratryl alcohol serves as a redox mediator for LP. In this mechanism, the oxidation to
the cation radical, followed by diffusion into the wood matrix, delivers the oxidizing power of the heme active site to the distal substrate lignin. Consistent with this hypothesis, in vitro
studies by Hammel et al. (31) showed maximal
depolymerization of lignin by LP occurred only in the presence of
veratryl alcohol. This hypothesis, however, has been questioned by
kinetic studies (18) and by chemical studies showing the chemical
instability of the veratryl alcohol cation radical (19).
Relevant to the question of the role of LP in lignin degradation is
whether LP can directly interact with the lignin polymer. To date,
crystallographic and spectroscopic data have yielded no information on
the substrate-binding site. Based on mainly theoretical studies at
least three substrate-binding sites have been proposed. Poulos et
al. (8) suggested a binding site for veratryl alcohol is an
access channel analogous to that found in CcP. This channel provides a
hydrophobic environment near to the heme. This channel allows for
direct interaction between the substrate and the heme. Modeling of
substrates into this channel indicates that only monomeric substrates
would bind or that larger linear polymers could bind through an
"end-on" manner. The ability of LP to directly interact with lignin
would not exclude this site, because the polymer could be envisioned to
bind through an exo mechanism. In addition to the heme access
channel, two long-range electron transfer routes have been proposed.
Schoemaker and Piontek (32) proposed residues 82-84 (His, Pro, Asn) of isozyme H8, whereas His82 was proposed also as the part of
the heme access channel (8). The other site was proposed by Blodig
et al. (20) involving residues Trp171 and
Leu172. Both of these long range sites connect the heme to
surface residues thus allowing for direct contact with lignin. Here, LP
could be envisioned to interact in an endo manner with lignin.
To gain insight into the nature of the binding site, we have
synthesized a larger (tetrameric) model of lignin to map the size of
the LP site of electron transfer. We utilized a tetrameric substrate,
which is actually larger than a tetramer, because a benzyl group,
rather than a methyl group, is attached to the terminal phenolic
oxygen. The tetramer was designed such that if the site of electron
transfer is the heme access channel, it could only gain access from an
end-on manner. This is clearly illustrated in Fig. 1 where the size of
the tetramer is compared with that of LP and its heme access channel.
Our studies clearly indicate, through time course studies and through
single turnover studies, that LP is able to oxidize the tetramer to
yield the dimer as the first product. In all incubations with the
tetramer, the monomeric, dimeric, trimeric, and tetrameric aldehydes
were detected. Of significance here is the finding that dimeric
products are formed from single turnover studies. This clearly maps the
site of electron transfer to be relatively unhindered and that LP can
oxidize the lignin polymer directly. In accordance with this
interpretation is our results from the chemical oxidation system. The
single electron oxidant, Ce4+, yielded the same profile of
products. Thus, LP can act as an endo lignin-degrading enzyme. This is
consistent with LP being able to interact with lignin directly and
catalyze C
-C
cleavage anywhere along the polymer.
Indirect evidence for interaction between LP and lignin (synthetic
polymer dehydropolymer) was recently obtained with a resonant mirror
biosensor by Johjima et al. (33). The interaction between the enzyme and dehydropolymer was only specific for LP; none was observed with laccase, MnP, or horseradish peroxidase. In addition to
this spectroscopic evidence, these workers also obtained rate constants
between LP compound I and the dehydropolymer. However, this study did
not reveal the nature of the products or reveal the location of the
substrate-binding site. Terminal phenolic groups may have served as the
reducing agent for both compounds I and II in this study.
Despite the finding that LP can directly interact with a large
polymeric lignin substrate, we also investigated whether veratryl alcohol could enhance the oxidation. As found in the oxidation of many
other substrates for LP, veratryl alcohol stimulated the oxidation of
the tetrameric model. The stimulation was measured to be ~20%, a
result which neither supported nor refuted the mediation hypothesis.
Past work has shown that facilitation of oxidation of secondary
substrates can be explained by several mechanisms. Although our
experiments were not designed to address the mediation issue, they are
more consistent with veratryl alcohol not acting as a mediator. In
incubations where veratryl alcohol acts as a mediator, a lag phase in
its oxidation has been observed (18). As shown in Fig. 5, a lag phase
of veratraldehyde formation was not observed. On the contrary, both the
tetramer and veratryl alcohol were oxidized simultaneously. Thus, it
most probably acted to protect LP from inactivation. In the case of
poor LP substrates, veratryl alcohol has been demonstrated to promote
the completion of the enzymatic catalytic cycle, decreasing the
inactivation of LP, and to consequently increase the oxidation rate of
the other substrate. The mechanism of stimulation was not further investigated. Even if some mediation does occur with veratryl alcohol,
these results do not refute our finding that LP is able to directly
oxidize large lignin molecules.
Our studies have also identified the location of the electron transfer
for polymeric substrates. We have made mutations among the amino acids
in the heme excess channel (24). Mutation of Glu146 at this
site was further characterized in the present study. This amino acid
residue was proposed to participate in veratryl alcohol binding and to
be involved in pH dependence (8). The enzyme mutated at the heme access
channel was able to oxidize the tetrameric lignin model resulting in
the same products as those by the wild type. Recently
Trp171 of the surface-exposed potential binding site was
mutated (28). The involvement of this amino acid in the oxidation of
veratryl alcohol was proposed. The W171S mutant, in contrast, was not
able to oxidize the tetramer, and no products were found. These
findings illustrate the significance of this site for electron transfer with veratryl alcohol and the polymeric lignin. The involvement of
Trp171 in veratryl alcohol oxidation is supported by other
studies (34, 35). The significance of Trp171 is also
suggested from the results on bifunctional MnPs. Certain MnPs from
Bjerkandera sp. and Pleurotus spp. oxidize
veratryl alcohol (36, 37). These enzymes also contain a Trp at the homologous site (37). Moreover, when MnP H4 was mutated to contain a
Trp (Trp168) (34, 35), it was able to oxidize veratryl alcohol.
In conclusion, our results support the hypothesis that LP can directly
oxidize lignin. Therefore, LP may have important role in the
degradation of phenolic and nonphenolic lignin. Site-directed mutagenesis studies suggest that the substrate-binding site is surface-exposed and that this site may accommodate both lignin and
veratryl alcohol. However, other compounds such as ABTS and DFAD may
bind at an alternate site. The presence of more than one binding site
is supported by the fact that the mutation of Trp171 in LP
did not affect the oxidation of certain dyes such as ABTS and DFAD
(28).