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

Article abstract of DOI:10.1074/jbc.M112.356378

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

Full text of DOI:10.1074/jbc.M112.356378

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2012/03/21/M112.356378.DC1.html
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 20, pp. 16903–16916, May 11, 2012
Published in the U.S.A.
Lignin-degrading Peroxidases from Genome of Selective
□
S
Ligninolytic Fungus Ceriporiopsis subvermispora*
Received for publication, February 25, 2012, and in revised form, March 19, 2012 Published, JBC Papers in Press, March 21, 2012, DOI 10.1074/jbc.M112.356378
Elena Fernández-Fueyo‡1, Francisco J. Ruiz-Dueñas‡2, Yuta Miki‡, María Jesús Martínez‡, Kenneth E. Hammel§3,
and Angel T. Martínez‡4
From the ‡Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, E-28040
Madrid, Spain and the §Forest Products Laboratory, United States Department of Agriculture, Madison, Wisconsin 53726
Background: The first genome of a selective lignin degrader is available.
Results: Its screening shows 26 peroxidase genes, and 5 genes were heterologously expressed and the catalytic properties
investigated.
Conclusion: Two new peroxidases oxidize simple and dimeric lignin models and efficiently depolymerize lignin.
Significance: Although lignin peroxidase and versatile peroxidase had not been reported in C. subvermispora, the genomic
screening suggests the presence of related lignin-degrading enzymes.
The white-rot fungus Ceriporiopsis subvermispora deligni- sical LiPs and VPs. These results offer new insight into selec-
fies lignocellulose with high selectivity, but until now it has
appeared to lack the specialized peroxidases, termed lignin
peroxidases (LiPs) and versatile peroxidases (VPs), that are
generally thought important for ligninolysis. We screened
the recently sequenced C. subvermispora genome for genes
that encode peroxidases with a potential ligninolytic role. A
total of 26 peroxidase genes was apparent after a structural-
functional classification based on homology modeling and a
search for diagnostic catalytic amino acid residues. In addi-
tion to revealing the presence of nine heme-thiolate peroxi-
dase superfamily members and the unexpected absence of the
dye-decolorizing peroxidase superfamily, the search showed
that the C. subvermispora genome encodes 16 class II
enzymes in the plant-fungal-bacterial peroxidase superfam-
ily, where LiPs and VPs are classified. The 16 encoded
enzymes include 13 putative manganese peroxidases and one
generic peroxidase but most notably two peroxidases con-
taining the catalytic tryptophan characteristic of LiPs and
VPs. We expressed these two enzymes in Escherichia coli and
determined their substrate specificities on typical LiP/VP
substrates, including nonphenolic lignin model monomers
and dimers, as well as synthetic lignin. The results show that
the two newly discovered C. subvermispora peroxidases are
functionally competent LiPs and also suggest that they are
phylogenetically and catalytically intermediate between clas-
tive lignin degradation by C. subvermispora.
Most of the carbon fixed by land photosynthesis is stored in
the plant polymers cellulose, hemicellulose, and lignin. Lignin is
an aromatic macromolecule (1) that protects the other two
polysaccharidic polymers against biodegradation. After plant
death, only certain specialized filamentous fungi, especially the
highly active basidiomycetes termed white-rot fungi (2), are
able to degrade lignin via oxidative mechanisms. Lignin biodeg-
radation is a key step in terrestrial carbon recycling, enabling
subsequent degradation of plant polysaccharides by complex
microbial communities. Its removal is also a central issue for
the current industrial use of lignocellulosic biomass (e.g. in cel-
lulose production for paper) as well as in lignocellulose biore-
fineries for the sustainable production of fuels, chemicals, and
materials from plant feedstocks (3). Industrial removal of lignin
often involves harsh chemical treatments, but lignin-degrading
fungi and their enzymes may present environmentally friendly
alternatives (4).
Two decay patterns are produced by white-rot fungi, being
characterized by degradation of both lignin and polysaccha-
rides (simultaneous decay) and by a preferential removal of
lignin leaving most of the polysaccharides unaffected (selective
decay) (5). Among selective decay basidiomycetes, Ceriporiop-
sis (Gelatoporia) subvermispora has been selected for delignifi-
cation of different types of wood (6–8) in paper pulp manu-
facture (9). More recently, it has also been assayed for
delignification in bioethanol production (10). The usual model
fungus in lignin biodegradation studies, Phanerochaete chrys-
osporium (11), is of limited interest for such applications
because it generally causes a simultaneous decay pattern (2).
From the first pilot scale trials on wood biopulping with C.
subvermispora, a high interest in its lignocellulolytic enzymatic
machinery has been sustained, with the double purpose of
enhancing enzyme production and discovering new or more
efficient enzymes of interest as industrial biocatalysts.
* This work was supported by the RAPERO Grant BIO2008-01533 and HIPOP
Grant BIO2011-26694 from the Spanish Ministry of Economy and Compet-
itiveness (to A. T. M. and F. J. R.-D., respectively), by PEROXICATS European
Project Grant KBBE-2010-4-265397 (to A. T. M.), and by United States
Department of Energy Grant DE-AI02-07ER64480 (to K. E. H.).
□
S
This article contains supplemental Figs. S1 and S2 and Table S1.
1 SupportedbyaJuntaparaAmpliacióndeEstudiosfellowshipoftheConsejo
Superior de Investigaciones Científicas, co-funded by the European Social
Fund.
2 SupportedbyaMinistryofEconomyandCompetitiveness(MINECO)Ramón
y Cajal contract.
3 To whom correspondence may be addressed. Tel.: 608-231-9528; Fax: 608-
231-9262; E-mail: kehammel@wisc.edu.
4 To whom correspondence may be addressed. Tel.: 34-918373112; Fax:
34-915360432; E-mail: ATMartinez@cib.csic.es.
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From Genome Screening to Demonstration of Ligninolysis
White-rot fungi secrete multiple isoenzymes of three heme heterologously (together with other C. subvermispora and ref-
peroxidases with proposed roles in oxidative ligninolysis as erence class II peroxidases) and have estimated their kinetic
follows: lignin peroxidases (LiPs),5 manganese peroxidases
(MnPs), and hybrid enzymes known as versatile peroxidases
(VPs) that combine the structural-functional properties of LiPs
and MnPs. In addition, these fungi secrete various oxidases to
produce the H2O2 required by peroxidases, and most of them
also secrete laccases (12). LiPs and VPs are able to oxidize and
cleave the recalcitrant nonphenolic structures that compose
the bulk of lignin, as shown by studies with dimeric lignin
model compounds (13, 14), and also by depolymerization
experiments with synthetic lignins in the case of LiPs (15).
MnPs and VPs oxidize Mn2ϩ to Mn3ϩ, which can oxidize only
the minor phenolic units in lignin (16). Likewise, laccases are
directly able to oxidize only the phenolic units in lignin (17).
Because the cleavage of nonphenolic lignin structures is cen-
tral to efficient ligninolysis, it might be expected that LiPs
and/or VPs are essential participants in the process. However,
among the above oxidoreductases, only laccase and MnP isoen-
zymes have been found in C. subvermispora cultures (18, 19),
although DNA hybridization/amplification with LiP sequences
was reported in initial studies (20, 21), and some manganese-
independent peroxidase activity has been detected (22). The
unexpected lack of LiPs or VPs in this selective delignifier has
led to various alternative hypotheses. One is that lipid peroxi-
dation reactions catalyzed by MnP might generate fatty acid-
derived oxyradicals that cleave lignin (23). There is some exper-
imental support for this mechanism, as it can depolymerize
nonphenolic synthetic lignin in vitro, but the reaction is slow
and inefficient (23–25). Alternatively, the ability of laccases to
oxidize nonphenolic lignin structures in the presence of various
low molecular weight naturally occurring redox mediators has
been demonstrated (26). This might lead to ligninolysis if the
fungus produces such mediators as extracellular metabolites or
lignin degradation products, but so far this has not been
demonstrated.
constants using high and low redox potential substrates. Fur-
thermore, we have assessed the ligninolytic capability of these
two peroxidases in experiments with a nonphenolic lignin
model dimer and with synthetic lignin.
MATERIALS AND METHODS
Fungal Strain and Genome Sequencing—The genome of
monokaryotic C. subvermispora strain B (31) was sequenced at
the Joint Genome Institute using a shotgun approach (39.0 Mb
total) in a project coordinated by Daniel Cullen (United States
Department of Agriculture Forest Products Laboratory, Madi-
son, WI) and Rafael Vicuña (Pontificia Universidad Católica de
Chile, Santiago, Chile). The results from gene prediction and
annotation are available for searching at the Joint Genome
Institute genome portal.
Genome Screening and Analysis of Peroxidase Models—The
process to obtain the complete inventory of C. subvermispora
heme peroxidase genes consisted of the following steps: (i)
screening the automatically annotated genome at the Joint
Genome Institute genome portal; (ii) revising and manually
curating (when necessary) the positions of introns, and the N
and C termini of the selected models; (iii) comparing the amino
acid sequence identities with related proteins; and (iv) confirm-
ing the presence of characteristic residues at the heme pocket
and substrate oxidation sites, after homology modeling using
crystal structures of related proteins from the RCSB Protein
Data Bank as templates.
Three MnP genes previously cloned from C. subvermispora
(GenBankTM accession numbers AAB03480, AAD45725, and
AAO61784 and described as MnP1, MnP3, and MnP4, respec-
tively) (32, 33) were identified (as models 116608, 139965, and
94398, respectively, showing 98–100% identities with the cor-
responding GenBankTM sequences). C. subvermispora MnP2
(GenBankTM entries AAB92247 and AAD43581 with 99%
sequence identity) was not localized in the genome but could be
an allelic variant of gene 50297 (94% identity). The deduced
amino acid sequences of the C. subvermispora heme peroxidase
gene models were compared with all the basidiomycete heme
peroxidase sequences (from GenBankTM and genomes) avail-
able to date (a total of 376 sequences, prokaryotic class I per-
oxidases excluded), and phylogenetic trees were constructed
using Poisson-corrected distances and unweighted pair group
method with arithmetic mean clustering (bootstrap consensus
trees were inferred from 1000 replicates).
The recent sequencing of the C. subvermispora genome at
the Joint Genome Institute, Department of Energy, allows a
new look at this longstanding problem. This project supple-
ments the first white-rot (27) and brown-rot (28) fungal
genome sequences to be obtained and reflects strong current
interest in lignocellulose decay mechanisms that may inspire
new biotechnological applications. In this study, an inventory
of all the peroxidase genes, with special emphasis on class II
heme peroxidases (29), was obtained with manual curation.
Then a structural-functional annotation of the predicted pro-
teins was produced, as recently reported for the Pleurotus
ostreatus genome (30), and their evolutionary history was
established by comparison with all the basidiomycete peroxi-
dase sequences currently available. Surprisingly, this annota-
tion revealed the presence in C. subvermispora of two genes
that encode putative LiP/VP enzymes. To assess the signifi-
cance of this finding, we have expressed these two peroxidases
The programs used in the above analyses include the follow-
ing: (i) BLAST (Basic Local Alignment Tool) at the National
Center for Biotechnology Information for searching nucleotide
and protein databases; (ii) MEGA5 (34) for conducting auto-
matic and manual sequence alignment during the heme perox-
idase gene search and curation, and for inferring evolutionary
trees; and (iii) SignalP 3.0 (35) for predicting the presence and
location of signal peptides and cleavage sites. The latter predic-
tions were validated by multiple alignment (from MEGA5) of
the 196 class II sequences available, including proteins whose N
termini have been sequenced. Theoretical molecular models
5 The abbreviations used are: LiP, lignin peroxidase; DHP, dehydrogenation
(lignin) polymer; GPC, gel permeation chromatography; HTP, heme-thio-
late peroxidase; MnP, manganese peroxidase; VP, versatile peroxidase;
ABTS, 2,2Ј-azinobis(3-ethylbenzothiazoline-6-sulfonate).
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From Genome Screening to Demonstration of Ligninolysis
were generated at the Swiss-Model automated protein homol- pH values) by the above E. coli-expressed peroxidases were
ogy-modeling server (36).
recorded in a Thermo Scientific Biomate5 spectrophotometer
(using ϳ0.01 ␮M enzyme). Oxidation of Mn2ϩ was followed at
pH 5 for the formation of the Mn3ϩꢀtartrate complex (⑀238 6.5
mMϪ1ꢀcmϪ1). Veratryl alcohol oxidation was followed at pH 3
for veratraldehyde (⑀310 9.3 mMϪ1ꢀcmϪ1) formation. Reactive
Black 5, 2,2Ј-azinobis(3-ethylbenzothiazoline-6-sulfonate)
(ABTS), and 2,6-dimethoxyphenol oxidation were all assayed at
pH 3.5. These reactions were monitored for Reactive Black 5
disappearance (⑀598 30 mMϪ1ꢀcmϪ1), for ABTS cation radical
formation (⑀436 29.3 mMϪ1ꢀcmϪ1), and for dimeric coeruli-
gnone formation (⑀469 5.5 mMϪ1 cmϪ1), respectively. All reac-
tions were at 25 °C and were initiated by addition of 0.1 mM
H2O2. ABTS and 2,6-dimethoxyphenol oxidation by some C.
subvermipora peroxidases shows double kinetics with sigmoi-
dal activity curves (at increasing substrate concentrations) that
enable calculation of two sets of kinetic constants (for high and
low efficiency oxidation sites), as reported previously for VP
(40, 41).
Gene Synthesis and Directed Mutagenesis—The mature pro-
tein-coding sequences of five selected C. subvermispora perox-
idase genes (49863, 99382, 112162, 118677, and 117436 models)
after the above manual curation, two VP genes from Pleurotus
eryngii (GenBankTM AF007244) and Pleurotus ostreatus
(genome 137757), and a P. chrysosporium LiP gene (Gen-
BankTM Y00262) were synthesized by ATG:biosynthetics
(Merzhausen, Germany) after verifying that all the codons had
been previously used for expressing other genes in the same
Escherichia coli strains (and substituting them when required).
The E183K mutation was introduced in C. subvermispora
gene 99382 by PCR using the expression plasmid pFLAG1-
99382 (see below) as template, and the QuikChange kit from
Stratagene. The 5Ј-CGCGGCTGCCGACAAGGTCGATC-
CTACCATCCCG-3Ј direct (mutated triplet underlined) and
reverse primers were synthesized. The PCR (50-␮l volume) was
carried out in a PerkinElmer Life Sciences GeneAmp PCR Sys-
tem 240 using 20 ng of template DNA, 500 ␮M each dNTP, 125
ng of each primer, 2.5 units of PfuTurbo DNA polymerase
(Stratagene), and the manufacturer’s buffer. Reaction condi-
tions were as follows: (i) a “hot start” at 95 °C for 1 min; (ii) 18
cycles at 95 °C for 50 s, 55 °C for 50 s, and 68 °C for 10 min; and
(iii) a final cycle at 68 °C for 10 min.
Means and standard errors for affinity constant (Km) and
enzyme turnover (kcat) values were obtained by nonlinear least
squares fitting of the experimental measurements to the
Michaelis-Menten model. Fitting of these constants to the nor-
malized equation v ϭ (kcat/Km)[S]/(1 ϩ [S]/Km) yielded the cat-
alytic efficiency values (kcat/Km) with their corresponding
standard errors.
E. coli Expression of Heme Peroxidase Genes—The five C.
subvermispora peroxidases, the P. eryngii and P. ostreatus VPs,
and the P. chrysosporium LiP mature protein-codifying
sequences were cloned in the expression vectors pFLAG1
(International Biotechnologies Inc.) or pET23a(ϩ) (Novagen).
The resulting plasmids pET23a-49863, pFLAG1-99382,
pET23a-112162, pFLAG1-118677, pET23a-117436, pFLAG1-
AF007244, pFLAG1-137757, and pFLAG1-Y00262 were
directly used for expression, and for the above-described
directed mutagenesis (pFLAG1-99382). E. coli DH5␣ was used
for plasmid propagation.
p-Dimethoxybenzene and Ferrocytochrome c Oxidation—Spec-
tral changes during oxidation of p-dimethoxybenzene (0.2 mM)
and ferrocytochrome c (15 ␮M) by the above peroxidases (ϳ0.1
␮M) at 25 °C in the presence of 0.1 mM H2O2 were recorded with
an Agilent 8453 diode-array spectrophotometer, showing the
formation of p-benzoquinone and ferricytochrome c, respec-
tively. Ferrocytochrome c was prepared before use by reducing
ferricytochrome c with sodium dithionite, followed by removal
of excess dithionite on a Sephadex G-25 column. p-Benzoqui-
none formation was followed at 245 nm in 20 mM sodium suc-
cinate (pH 3.5). Decrease of ferrocytochrome c concentration
was followed at 550 nm in 20 mM sodium succinate (pH 4).
Controls without enzyme and without H2O2 were included.
Oxidative Degradation of a Lignin Model Dimer—Unlabeled
and ring-14C-labeled (1.0 mCi mmolϪ1) 4-ethoxy-3-methoxy-
phenylglycerol-␤-guaiacyl ether were prepared, and their
erythro and threo isomers were chromatographically separated,
as described earlier (42).
The different peroxidases (and the 99382 E183K variant)
were produced in E. coli W3110 (pFLAG1-derived plasmids)
and BL21(DE3)pLysS (pET23a-derived plasmids). Cells were
grown for 3 h in Terrific Broth (37), induced with 1 mM isopro-
pyl ␤-D-thiogalactopyranoside, and grown further for 4 h. The
apoenzyme accumulated in inclusion bodies, as observed by
SDS-PAGE, and was solubilized using 8 M urea. Subsequent in
vitro refolding of C. subvermispora peroxidases and Pleurotus
VPs was performed using 0.16 M urea, 5 mM Ca2ϩ, 20 ␮M hemin,
0.5 mM oxidized glutathione, 0.1 mM dithiothreitol, and 0.1
mg/ml protein, at pH 9.5 (38). C. subvermispora peroxidase
112162 was refolded using 1 M urea, 5 mM Ca2ϩ, 10 ␮M hemin,
0.5 mM oxidized glutathione, 0.1 mM dithiothreitol, and 0.1
mg/ml protein, at pH 9. Refolding of P. chrysosporium LiP was
performed using 2.1 M urea, 5 mM Ca2ϩ, 10 ␮M hemin, 0.7 mM
oxidized glutathione, 0.1 mM dithiothreitol, and 0.2 mg/ml pro-
For reactions involving product analysis, the radiolabeled
erythro or threo dimer was treated either with one of the two C.
subvermispora LiP-like peroxidases (from genes 118677 and
99382) or with P. chrysosporium LiPH8 in 10 mM sodium ace-
tate (pH 3.0) at 25 °C for 1 h. The products formed were ana-
lyzed by reversed phase high performance liquid chromatogra-
phy (HPLC) using a Gilson system equipped with a C-18
column (Phenomenex Luna C18(2); 150 by 4.6 mm, 5-␮m par-
ticle size), and methanol/water as mobile phase (35:65 for 15
min, followed by 50:50) at a flow rate of 1 mlꢀminϪ1. Elution was
monitored at 255 nm, and the 14C content in collected fractions
(0.5 ml) was measured in a liquid scintillation counter. HPLC in
tein, at pH 8 (39). Active enzyme was purified by Resource-Q
Ϫ1
chromatography using a 0–300 mM NaCl gradient (2 mlꢀmin
,
20 min) in 10 mM sodium tartrate (pH 5.5) containing 1 mM
CaCl2.
Kinetic Constants on Selected Substrates—Absorbance
changes during substrate oxidation in 0.1 M tartrate (at various conjunction with gas chromatography/mass spectrometry was
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From Genome Screening to Demonstration of Ligninolysis
used in parallel analyses with unlabeled dimers to confirm the
identity of the products obtained.
For the class II peroxidases we found (numbers 1–16 supple-
mental Table S1), the classification was based on the presence/
absence in the molecular models (obtained using crystal struc-
tures of related enzymes as templates) of the following: (i) an
exposed tryptophan (two models) putatively involved in oxida-
tion of veratryl alcohol and other high redox potential aromat-
ics (Wc, homologous to Trp-171 in P. chrysosporium LiP isoen-
zyme-H8 and to Trp-164 in P. eryngii VP isoenzyme-VPL) (Fig.
1, A and B); and/or (ii) a putative Mn2ϩ oxidation site (14 mod-
els) formed by three acidic residues near the most internal
heme propionate (Ea, Eb, and Ec, homologous to P. chrysospo-
rium MnP1 Glu-35, Glu-39, and Asp-179, and to P. eryngii VPL
Glu-36, Glu-40 and Asp-175, respectively) (Fig. 1, B and D).
The only C. subvermispora peroxidase showing both of the
above two catalytic sites (gene 99382) was initially classified as a
putative VP (Fig. 1B). The 13 models showing only the Mn2ϩ
oxidation site (genes 1–13 in supplemental Table S1) were clas-
sified as likely MnPs (Fig. 1D). The single peroxidase containing
only the catalytic tryptophan (gene 118677) was classified as a
putative LiP (Fig. 1A). Finally, the model lacking both oxidation
sites (gene 112162) was classified as a generic peroxidase (Fig.
1C), related to the Coprinopsis cinerea peroxidase (44).
Among the MnPs, two types were identified (see supplemen-
tal Table S1). (i) MnP-long, represented by 12gene models in the
C. subvermispora genome, resembles typical MnPs described in P.
chrysosporium, whose amino acid sequence is longer than those of
LiPs and VPs and includes an additional disulfide bridge. (ii) MnP-
short, represented by only one gene model (gene 124076), resem-
bles some Phlebia radiata MnPs. MnP-long includes five “extra
long” MnPs, whose differential catalytic properties are still to be
reported.
For experiments to determine reaction kinetics, unlabeled
erythro or threo dimer was treated with C. subvermispora
peroxidase at 25 °C in the same buffer. Approximate turn-
over numbers (sϪ1) at various dimer concentrations were
obtained from the increase in absorbance at 310 nm due to
benzylic carbonyl formation, i.e. production of 4-ethoxy-3-
methoxybenzaldehyde, 1-(4-ethoxy-3-methoxyphenyl)-3-
hydroxy-2-(2-methoxyphenoxy)-propan-1-one, and 1-(4-
ethoxy-3-methoxyphenyl)-2,3-dihydroxypropan-1-one (⑀310
9 mMϪ1ꢀcmϪ1). Kinetic constants were then calculated as des-
cribed above.
Enzymatic Depolymerization of Synthetic Lignin—A radiola-
beled syringyl/guaiacyl dehydrogenation polymer (DHP) with a
syringyl/guaiacyl ratio of ϳ4:1 was prepared by enzymatic
Ϫ1
copolymerization of ␤-[14C]sinapyl alcohol (0.01 mCi mmol
)
and unlabeled coniferyl alcohol using horseradish peroxidase as
described previously (43). The synthetic lignin was then frac-
tionated on a column of Sephadex LH-20 as described (43), and
the high molecular mass fractions (approximately Ն21 kDa)
were pooled for use in depolymerization experiments.
Enzymatic depolymerization of the DHP by the same peroxi-
dases used in the dimer degradation assays was investigated in
10 mM sodium acetate (pH 4.5) containing 0.25% Tween 20,
1.5 ϫ 104 dpm (188 ␮g) DHP, and 0.01 ␮M enzyme in a final
volume of 40 ml, in the presence or absence of 10 mM veratryl
alcohol. Reactions were conducted at 25 °C by adding H2O2 (7.5
mM final concentration in experiments with veratryl alcohol
and 0.3 mM in experiments without veratryl alcohol) continu-
ously over 24 h with a syringe pump (15). Control reactions
without enzyme were run for comparison. The reaction mix-
tures were then concentrated by rotary vacuum evaporation,
redissolved in N,NЈ-dimethylformamide containing 0.1 M LiCl,
and centrifuged as described earlier (15). Molecular mass dis-
tributions of the supernatant fractions were assessed by gel per-
meation chromatography (GPC) on a 1.8 ϫ 30-cm column of
Sephadex LH20, using N,NЈ-dimethylformamide containing
0.1 M LiCl as the mobile phase. Fractions (2 ml) were collected
and assayed for 14C in a liquid scintillation counter (15).
Chemicals—p-Dimethoxybenzene, N,NЈ-dimethylform-
amide, dithiothreitol, 2,6-dimethoxyphenol, ferricytochrome
c, hemin, isopropyl ␤-D-thiogalactopyranoside, lithium chlo-
ride, manganese sulfate, methanol, oxidized glutathione, Reac-
tive Black 5, sodium dithionite, sodium tartrate, Tween 20,
veratryl alcohol, and other unlabeled chemicals were purchased
from Sigma, with the exception of H2O2 that was from Merck,
and ABTS that was from Roche Applied Science.
An additional single model (supplemental Table S1, gene
83438) has predicted protein folding and heme pocket architec-
ture similar to that of the above class II peroxidases. However, it
is a class I (of prokaryotic origin but in the same superfamily)
cytochrome c peroxidase and is therefore not included in the
subsequent comparisons.
The other nine models (numbers 18–26 in supplemental
Table S1) correspond to putative peroxidases from the HTP
superfamily and are most probably related to the Agrocybe
aegerita peroxygenase (GenBankTM entries CAV28568 and
CAV28569) (45). This superfamily is characterized by the pres-
ence in the molecular structures (that of gene 80799 is shown in
Fig. 2A) of a cysteine residue acting as the fifth heme ligand,
instead of the histidine residue present in the above peroxi-
dases, among other structural details (Fig. 2B).
Catalytic Properties of Peroxidases Expressed in E. coli—Among
the 15 class II genes in the C. subvermispora genome, including
genes putatively encoding ligninolytic peroxidases, we selected
118677, 99382, 117436, 49863, and 112162 for heterologous
expression, as representative for the main families identified.
Our first goal was to investigate their catalytic properties on
selected peroxidase substrates and in this way to confirm or
modify their putative structural/functional classifications.
Additionally, we aimed to evaluate their ability to degrade
lignin using a simple dimeric model compound and a polymeric
synthetic lignin (DHP).
RESULTS
Inventory of Heme Peroxidase Genes in the C. subvermispora
Genome—Among the 12,125 gene models identified in the C.
subvermispora genome distributed in 740 main scaffolds, 26
models were found to encode heme peroxidases after manual
revision of the possible candidates from automatic annotation
(supplemental Table S1 provides the reference, scaffold, posi-
tion, best hit, amino acid sequence identity, and peroxidase
types encoded by these genes).
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FIGURE 1. Homology models for the molecular structures of four hypothetical members of class II (fungal peroxidases) from the C. subvermispora
genome. A, 118677 peroxidase; B, 99382 peroxidase; C, 112162 peroxidase; and D, 117436 peroxidase. The general folding is shown, together with details of
the heme cofactor and residues forming the substrate oxidation sites of MnP/VP (two glutamates and one aspartate, B and D) and LiP/VP (exposed tryptophan,
A and B)-type enzymes, as well as the homologous residues in the other proteins (residues and cofactor as Corey-Pauling-Koltun (CPK), colored sticks). The
crystal structures of P. eryngii VP (Protein Data Bank code 2BOQ) and P. chrysosporium MnP (Protein Data Bank code 3M5Q) were used as templates. The amino
acid numbering refers to putative mature sequences, after manual curation of their signal peptide sequences.
FIGURE 2. Homology model for the molecular structure of a member of the HTP superfamily from the C. subvermispora genome. The general folding of
a putative C. subvermispora HTP (model 80799) is shown (A) together with details of its heme region (B, residues and cofactor as CPK-colored sticks, and metal
ion as a blue van der Waals sphere). The crystal structure of L. fumago chloroperoxidase (Protein Data Bank code 1CPO) was used as a template. The amino acid
numbering refers to putative mature sequence after manual curation of signal peptide sequences.
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From Genome Screening to Demonstration of Ligninolysis
After manual curation, which entailed revision of predicted
introns and N and C termini, we used the codifying DNA
sequences to express the mature proteins in E. coli (see supple-
mental Fig. S1, A–D for sequences). We used this expression
system because, although it requires heme insertion and pro-
tein refolding in vitro, the yields are high (ϳ200 mg of protein
per 5 liters of culture). Moreover, the lack of host-dependent
protein glycosylation in this prokaryotic expression system
facilitated comparisons of catalytic properties in our subse-
quent investigations. We purified the resulting active peroxi-
dases to electrophoretic homogeneity in one chromatographic
step using a Resource-Q column (Fig. 3). The average yield of
the refolding process was ϳ6 mg of pure active protein per each
200 mg of protein recovered from the inclusion bodies. The
molecular masses estimated by SDS-PAGE (Fig. 3, inset) coin-
FIGURE 3. Purification of the enzyme product of the putative C. subver-
cided with the values obtained from the mature protein
sequences, and electronic absorption spectra revealed the pres-
ence of Soret (406 nm) and other typical bands of resting state
peroxidases, thus confirming correct incorporation of the heme
cofactor (see the electronic absorption spectra in supplemental
Fig. S2). Moreover, the A410/A280 ratio was similar for all the
refolded enzymes, with an average value of 4.
mispora VP gene (99382) after E. coli expression and in vitro activation.
The gene (optimized manually curated codifying sequence) was synthesized,
cloned in pFLAG1, expressed in E. coli W3110, refolded in vitro, and purified
(38). The VP peak obtained using a Resource-Q column and a 0–300 mM NaCl
gradient is shown. The elution profiles at 280 nm (total protein) and 410 nm
(heme proteins) are shown by dotted and continuous lines, respectively (the
actual NaCl gradient, from conductivity measurement, is shown by the
dashed line). The electrophoretic homogeneity of the enzyme was confirmed
by SDS-PAGE, as shown in the inset (right lane), and a molecular mass of ϳ35
kDa was estimated.
Next, we characterized the substrate specificity of our heter-
ologously expressed peroxidases, including for comparison sev-
eral well characterized enzymes as follows: P. eryngii VP, a
closely homologous P. ostreatus VP, and P. chrysosporium
LiPH8. For this experiment we used the following: (i) two sim-
ple high redox potential substrates (veratryl alcohol and Reac-
tive Black 5), which can be oxidized only at the LiP or VP cata-
lytic tryptophan; (ii) two low redox-potential substrates (ABTS
and 2,6-dimethoxyphenol), which are oxidized at the above cat-
alytic tryptophan with high efficiency but are also oxidized at a
LiPH8 (ϳ5 sϪ1ꢀmMϪ1 versus ϳ90 sϪ1ꢀmMϪ1), it is clearly a LiP-
like enzyme.
The putative VP classification for model 99382 was not con-
firmed, as it was unable to oxidize Mn2ϩ (Table 1) despite hav-
ing the three acidic residues that form the typical Mn2ϩ oxida-
tion site in MnPs and VPs (Fig. 1B). This finding, together with
our observation that peroxidase 99382 oxidized veratryl alcohol
(Table 1), suggested that it, like peroxidase 118677, is an LiP-
like enzyme with a low catalytic efficiency on veratryl alcohol
(ϳ3 sϪ1ꢀmMϪ1). In agreement, the catalytic efficiencies of
enzyme 99382 for ABTS and 2,6-dimethoxyphenol oxidation at
high and low efficiency sites (Table 1) were similar to those we
found for the LiP-like peroxidase 118677, and its substrate
specificity resembles that of P. chrysosporium LiPs (although
typical LiPs lack the low efficiency site).
second site by VPs and generic peroxidases; and (iii) Mn2ϩ
,
which is oxidized only at the MnP or VP Mn2ϩ oxidation site
(Table 1). In this way, we were able to check each enzyme’s puta-
tive peroxidase classification as an LiP, VP, MnP, or generic per-
oxidase. The abovementioned existence of two oxidation sites for
some phenols and dyes has been reported for VP (40, 41), although
the second one is still to be fully characterized.
One peculiarity of the two C. subvermispora LiP-like
enzymes, as compared with P. chrysosporium LiPs, is their abil-
ity to directly oxidize Reactive Black 5 (Table 1), a typical VP
substrate that LiP cannot oxidize in the absence of veratryl alco-
hol as a mediator. Moreover, as noted above, the catalytic effi-
ciencies of the two C. subvermispora LiP-like enzymes on vera-
tryl alcohol are low relative to values for typical LiPs. Instead,
they are similar to values found for typical VPs (ϳ2 sϪ1ꢀmMϪ1).
Accordingly, we surmise that C. subvermispora peroxidases
99382 and 118677 are VP-type enzymes that have lost the abil-
ity to oxidize Mn2ϩ and therefore must be formally classified as
LiPs. They appear as such in Tables 1 and supplemental Table
S1 and in Fig. 7.
As shown by the kinetic constants obtained (Table 1), the
putative MnP classification for model 117436 was confirmed by
its very high catalytic efficiency for Mn2ϩ oxidation (Ͼ5000
s
Ϫ1ꢀmMϪ1), its negligible efficiency (nonsaturation of the
enzyme prevented estimation of other kinetic constants) for
2,6-dimethoxyphenol oxidation at a low efficiency site (Ͻ0.05
s
Ϫ1ꢀmMϪ1), and its complete lack of activity in all the other
reactions investigated (and very similar results were obtained
for the 49863 peroxidase). The classification of peroxidase
112161 as a generic (nonligninolytic) peroxidase was also con-
firmed by its inability to oxidize veratryl alcohol and Mn2ϩ, as
well as Reactive Black 5, although it oxidizes ABTS and 2,6-
dimethoxyphenol (Table 1).
The lack of Mn2ϩ oxidation activity in the 118677 peroxidase
is explained by its having a serine in place of one of the three
acidic (glutamate or aspartate) residues required for Mn2ϩ oxi-
dation (Fig. 1, A and D). However, the lack of Mn2ϩ oxidation
activity by the 99382 peroxidase is more difficult to explain
Likewise, the putative LiP classification for model 118677
appears appropriate, as it exhibited no activity toward Mn2ϩ
but was able to oxidize the standard LiP substrate veratryl alco-
hol (Table 1). Although the catalytic efficiency of peroxidase
118677 is considerably lower than that of P. chrysosporium because it has the three necessary acidic residues (Fig. 1B) that
16908 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287•NUMBER 20•MAY 11, 2012

From Genome Screening to Demonstration of Ligninolysis
TABLE 1
Kinetic constants (Km (␮M), kcat (s؊1), and kcat/Km (s؊1ꢀmM؊1)) for C. subvermispora class II peroxidases (and the 99382 E183K variant) on the
simple substrates veratryl alcohol (VA), Reactive Black 5 (RB5), ABTS, 2,6-dimethoxyphenol (DMP), and Mn2؉, compared with typical LiP and VP
Kinetic constants were estimated at 25 °C in 0.1 M tartrate (pH 3) for veratryl alcohol, pH 3.5 for Reactive Black 5, 2,6-dimethoxyphenol and ABTS, and pH 5 for Mn2ϩ. GP
is generic peroxidase. Means and 95% confidence limits are shown. Dashes correspond to efficiency values (i.e. kcat/km ratios) where both kcat and km are 0.
C. subvermispora genome
99382
118677
99382
117436
112162
(GP)
(LiP1a)
(LiP2a)
(E183K)
(MnP10)
LiPb
VPc
VA
Km
kcat
3120 Ϯ 526
1620 Ϯ 290
2860 Ϯ 179
0
0
0
-
190 Ϯ 17
17.5 Ϯ 0.5
92.0 Ϯ 6.0
4130 Ϯ 320
8.6 Ϯ 0.7
8.7 Ϯ 0.6
8.40 Ϯ 0.37
0
9.5 Ϯ 0.2
k
cat/Km 2.8 Ϯ 0.3
5.4 Ϯ 0.7
2.9 Ϯ 0.4
-
2.3 Ϯ 0.1
RB5
Km
4.0 Ϯ 0.7
4.5 Ϯ 0.6
7.3 Ϯ 0.5
1620 Ϯ 138
4.6 Ϯ 0.6
7.2 Ϯ 0.4
1560 Ϯ 120
0
0
-
0
0
-
0
0
-
0
0
-
3.4 Ϯ 0.3
5.5 Ϯ 0.3
1310 Ϯ 90
3.0 Ϯ 0.2 (1090 Ϯ 42)
8.1 Ϯ 0.2 (186 Ϯ 3)
2700 Ϯ 140 (200 Ϯ 3)
kcat
9.8 Ϯ 0.9
kcat/Km 2460 Ϯ 185
ABTSd Km
4.0 Ϯ 0.4 (4250 Ϯ 508) 7.2 Ϯ 1.0 (NS)e
4.2 Ϯ 0.3 (4110 Ϯ 468)
40.5 Ϯ 5
23.0 Ϯ 1.6
kcat
9.3 Ϯ 0.2 (56 Ϯ 2)
11.3 Ϯ 0.5 (NS)
9.5 Ϯ 0.4 (61 Ϯ 3)
39.6 Ϯ 2.6 13.0 Ϯ 0.2
k
cat/Km 2300 Ϯ 200 (13 Ϯ 1)
1600 Ϯ 200 (3 Ϯ 0.1) 2260 Ϯ 236 (15 Ϯ 1)
976 Ϯ 16
563 Ϯ 36
DMPd Km
29 Ϯ 2 (NS)
37 Ϯ 4 (NS)
30 Ϯ 2 (NS)
NS
185 Ϯ 28
33.7 Ϯ 2
181 Ϯ 13
0
0
-
6 Ϯ 1
78 Ϯ 8 (10500 Ϯ 400)
kcat
6.4 Ϯ 0.1 (NS)
6.1 Ϯ 0.1 (NS)
6.9 Ϯ 0.1 (NS)
NS
10.0 Ϯ 0.1
56 Ϯ 1 (30 Ϯ 0)
k
cat/Km 222 Ϯ 13 (0.1 Ϯ 0)
167 Ϯ 17 (0.1 Ϯ 0)
235 Ϯ 15 (0.1 Ϯ 0)
Ͻ0.1
1720 Ϯ 153 71 Ϯ 6 (2.8 Ϯ 0.1)
Mn2ϩ
Km
0
0
-
0
0
-
9720 Ϯ 1530
135 Ϯ 8
59 Ϯ 9
0
0
-
181 Ϯ 10
kcat
331 Ϯ 20
5600 Ϯ 500
275 Ϯ 4
kcat/Km
14 Ϯ 0
1520 Ϯ 70
a LiP-VP transition enzymes.
b Data are from GenBankTM accession number Y00262 LiPH8.
c Data are from GenBankTM accession number AF007244 VPL.
d ABTS and DMP showed biphasic kinetics when oxidized by some VP-type and LiP-type enzymes enabling determination of two sets of constants (the second one, charac-
terized by a low catalytic efficiency, is shown in parentheses).
e NS, Km and kcat values were not determined because of nonsaturation of the enzyme, but catalytic efficiencies were estimated from slope of observed activity versus sub-
strate concentration.
are conserved in the Mn-oxidizing enzymes (Fig. 1D). The of the C. subvermispora peroxidases (model 118677) and the
explanation may lie in the presence of a fourth acidic residue Pleurotus VP (Fig. 4B).
(Glu-183 contiguous to conserved Asp-182) (Fig. 1B), which
Next, we assayed the ability of the heterologously expressed
constitutes a difference with respect to typical MnPs and VPs. C. subvermispora LiPs to cleave two nonphenolic lignin model
In support, the E183K variant of the 99382 peroxidase is able to dimers, the erythro and threo isomers of 4-ethoxy-3-methoxy-
oxidize Mn2ϩ, as shown in Table 1, albeit with lower catalytic phenylglycerol-␤-guaiacyl ether, which represent the principal
efficiency than typical MnPs and VPs, whereas its other cata- ␤-O-4 interunit linkage in lignin (1). The substrates were 14C-
lytic properties are not significantly modified.
labeled to facilitate detection and quantification of oxidized
Oxidation of Recalcitrant Substrates by C. subvermispora products by HPLC. The results (Fig. 5A) revealed that C. sub-
Peroxidases—LiPs and VPs, unlike other peroxidases, are able vermispora LiP 118677 oxidized the dimer (peak 1) to yield
to oxidize aromatics that lack strongly electron-donating –OH 4-ethoxy-3-methoxybenzaldehyde (peak 2), the uncleaved
or –NH2 substituents. This ability stems from their high redox ketone with an oxo group at C␣ (peak 3), and 1-(4-ethoxy-3-
potential and is central to their degradative activity on lignin. In methoxyphenyl)-2,3-dihydroxypropan-1-one (peak 4), together
addition, the ligninolytic activity of LiPs and VPs is likely pro- with small amounts of 1-(4-ethoxy-3-methoxyphenyl)glycerol
moted by their ability to oxidize bulky molecules due to the (peak 5). Similar reaction products were produced by the 99382
presence on their surface of an exposed tryptophan, which peroxidase (data not shown). The threo isomer was oxidized
serves to receive electrons from the solvent region and transfer with a higher kcat than the erythro isomer but was not cleaved
them to the heme cofactor.
To assess the reactivity of our heterologously expressed consisted of the uncleaved ketone 3 (Fig. 5, A and B).
enzymes on recalcitrant substrates, we began by performing Finally, we assessed the ability of the heterologously
more rapidly, as virtually all of the additional oxidized product
oxidations of two electron donors classically used to show the expressed C. subvermispora LiPs to depolymerize a radiola-
above properties, p-dimethoxybenzene and ferrocytochrome c. beled synthetic lignin (DHP). Fig. 6, A and B, shows the results
As shown in Fig. 4, where the same concentration of each of GPC analyses of lignin treated with C. subvermispora perox-
enzyme (0.1 ␮M) was used, the C. subvermispora MnP (model idase 118677 in the presence and absence of veratryl alcohol,
117436) was unable to oxidize either substrate, in agreement respectively, together with controls without enzyme. Similar
with the lack of a catalytic tryptophan. P. chrysosporium LiP GPC profiles were obtained for the 99382 peroxidase (data not
catalyzed the fastest oxidation of p-dimethoxybenzene to shown). These results show that the two C. subvermispora LiPs
p-benzoquinone, followed by the two C. subvermispora peroxi- partially depolymerized polymeric lignin, provided veratryl
dases (models 99382 and 118677) and the Pleurotus VP, which alcohol was present. For comparison, we show here the result
all exhibited similar oxidation rates (Fig. 4A). However, the P. obtained in this work using P. chrysosporium LiPH8 in the pres-
chrysosporium LiP did not catalyze the fastest oxidation of fer- ence of veratryl alcohol (Fig. 6C). We conclude that C. subver-
rocytochrome c, as slightly higher rates were observed for one mispora peroxidases 99382 and 118677 are fully competent
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From Genome Screening to Demonstration of Ligninolysis
FIGURE 4. Oxidation of p-dimethoxybenzene (A) and ferrocytochrome c
(B) by three class II peroxidases from the C. subvermispora genome com-
FIGURE 5. Oxidative degradation of a nonphenolic lignin model dimer
pared with oxidation by VP and LiP. p-Dimethoxybenzene oxidation to
p-benzoquinone by E. coli-expressed C. subvermispora 99382 (LiP), 118677
(LiP), and 117436 (MnP), P. ostreatus VP (genome 137757) and P. chrysospo-
rium LiPH8 (GenBankTM Y00262) was followed at 245 nm in the presence of
H2O2 (A). Ferrocytochrome c oxidation to ferricytochrome c was followed at
550nm(B). ControlswithoutenzymeandwithoutH2O2 wereincludedinboth
cases.
and estimation of the corresponding kinetic constants. A, erythro (contin-
uous line) and threo (dashed line) isomers of 14C-labeled 4-ethoxy-3-methoxy-
phenylglycerol-␤-guaiacyl ether (peak 1) were treated with E. coli-expressed
C. subvermispora LiP (gene 118677), and the completed reactions were ana-
lyzed by HPLC. The main products were 4-ethoxy-3-methoxybenzaldehyde
(peak 2), 1-(4-ethoxy-3-methoxyphenyl)-3-hydroxy-2-(2-methoxyphenoxy)-
propan-1-one (peak 3), and 1-(4-ethoxy-3-methoxyphenyl)-2,3-dihydroxy-
propan-1-one (peak 4). The minor peak 5 corresponds to small amounts of
erythro and threo 1-(4-ethoxy-3-methoxyphenyl)glycerol formed from the
erythro and threo dimers, respectively. B, different concentrations of
the erythro (continuous line) and threo (dashed line) isomers of the lignin
model dimer were treated with C. subvermispora LiP 118677. Turnover
numbers (sϪ1) were obtained at different dimer concentrations by spec-
trophotometric estimation of the main products formed, and the kinetic
constants were then calculated.
LiPs, despite their relatively low catalytic efficiency with the
standard LiP substrate veratryl alcohol.
Evolutionary Relationships of C. subvermispora and Other
Basidiomycete Peroxidases—Over 400 sequences of basidiomy-
cete heme peroxidases are available to date (from GenBankTM
and genomes). To establish the evolutionary relationships of C.
subvermispora peroxidases, a comparison of their deduced pro- ing all members (47 sequences), of the dye-decolorizing perox-
tein sequences with the other peroxidases from basidiomycetes idase superfamily (group D). As for the HTPs (a total of 133
was performed with MEGA5 (44 class I sequences were sequences), the nine C. subvermispora sequences are distrib-
excluded). In the dendrogram obtained (Fig. 7), all branches uted in three of the four main clusters defined in this superfam-
that do not include at least one C. subvermispora sequence are ily (Fig. 7, E, G, and H) with sequences 122198, 81391, and
collapsed for simplicity. This is the case for the branch, includ- 114787 forming a homogeneous subcluster in cluster E.
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From Genome Screening to Demonstration of Ligninolysis
clusters (including the 10 P. chrysosporium LiP genes) are
included, together with additional VP and LiP sequences from
other fungi. The position of these two C. subvermispora peroxi-
dases (118677 and 99382), grouping together in cluster A,
agrees with their LiP-like catalytic and lignin-degrading prop-
erties as described above, and thus with their description as new
lignin-degrading peroxidases occupying an intermediate posi-
tion between typical LiPs and VPs.
DISCUSSION
Overview—One of the most important pieces of evidence on
the central role of ligninolytic peroxidases in lignin degradation
comes from the comparison of the first white-rot (P. chrysospo-
rium) (27) and brown-rot (Postia placenta) (28) genomes to be
sequenced. Up to 15 ligninolytic peroxidase genes (10 lips and 5
mnps) were identified in the P. chrysosporium genome. By con-
trast, no such peroxidases are encoded in the genome of P.
placenta, a related species that degrades polysaccharides while
removing little of the lignin (4). These findings confirm earlier
genetic and biochemical studies with the two fungi and point to
a central role for LiPs and MnPs in efficient biological ligninoly-
sis (47, 48). VP genes substitute for LiP genes in some genomes,
such as that of P. ostreatus (30).
In light of these results, the apparent paucity of ligninolytic
peroxidases in C. subvermispora, a white rot fungus closely
related to P. chrysosporium, has been a perplexing research
problem. When the C. subvermispora genome project was ini-
tiated, four MnP isoforms were the only peroxidases previously
isolated (and/or cloned) from this organism (19, 32, 33, 49–51).
The genomic screening results we report here now expand this
number to include 26 heme peroxidase genes belonging to the
plant-fungal-bacterial peroxidase superfamily (17 genes) and
the HTP superfamily (nine genes) (29, 52). Surprisingly, no
members of the growing dye-decolorizing peroxidase super-
family were identified, although they seem widespread among
white-rot fungi (47 basidiomycete sequences available to date)
and can oxidize nonphenolic lignin model compounds (53).
The absence of related genes in C. subvermispora rules out their
involvement in lignin degradation by this fungus.
FIGURE 6. Lignin depolymerization by C. subvermispora LiP as compared
HTPs—The chloroperoxidase from the ascomycete Lep-
toxyphium fumago was the only known fungal HTP for years,
but the number of similar genes now apparent in basidiomy-
cetes has increased greatly after the sequencing of the A.
aegerita peroxygenase gene (45). Basidiomycete HTPs are
among the most versatile peroxidase types, able to catalyze a
wide variety of oxygenation and oxidation reactions on aro-
matic and aliphatic compounds (52, 54). However, the A.
aegerita peroxygenase fails to cleave synthetic lignin, although
with depolymerization by P. chrysosporium LiP. 14C-Labeled synthetic
lignin was treated with E. coli-expressed C. subvermispora LiP (gene 118677)
in the presence (A) and absence (B) of veratryl alcohol and with P. chrysospo-
rium LiPH8 (GenBankTM Y00262) in the presence of veratryl alcohol (C). Black
symbols indicate reactions with enzymes, and open symbols indicate controls
without enzymes. Total recoveries of initially added 14C from complete reac-
tions before GPC analysis were 72% for reaction A, 81% for reaction B, and
68% for reaction C. Recoveries from control reactions were somewhat higher
as reported earlier (15). The arrows indicate elution volumes of two polysty-
rene molecular mass standards (1800 and 500) and of veratryl alcohol (168).
The most interesting information for our purposes was it can oxidize nonphenolic lignin model dimers (55). It is also
obtained for the class II peroxidases (a total of 196 sequences) in unable to oxidize chloride efficiently, although it has bro-
the plant-fungal-bacterial peroxidase superfamily. Most of the moperoxidase activity (56), and for this reason it is unlikely to
C. subvermispora sequences in this group correspond to long cleave lignin through the agency of hypochlorous acid as the L.
and extra long MnPs (12 sequences) in cluster B, together with fumago chloroperoxidase has been shown to do (57). Accord-
all the related enzymes from P. chrysosporium and Dichomitus ingly, a role for C. subvermispora HTPs in ligninolysis appears
squalens, among other basidiomycetes. However, the unique unlikely.
MnP-short sequence and the two LiP-type sequences (from
Plant-Fungal-Bacterial Peroxidases—The C. subvermispora
genes 118677 and 99382) are included in cluster A, where inter- genome encodes a single class I cytochrome c peroxidase, a
mixed MnP-short and VP subclusters, as well as LiP/VP sub- mitochondrial enzyme of prokaryotic origin. It encodes numer-
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From Genome Screening to Demonstration of Ligninolysis
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From Genome Screening to Demonstration of Ligninolysis
FIGURE8. Detailofthecatalytictryptophansurfaceenvironment(7residues)intwo C. subvermisporaperoxidases(top)comparedwithtypicalVPand
LiP (bottom). A, C. subvermispora 118677 peroxidase (homology model) solvent access surface (showing the Asp-165, Gln-167, His-168, Trp-171, Glu-250,
Arg-260, Leu-264, and Ala-267). B, C. subvermispora 99382 peroxidase (homology model) solvent access surface (showing Thr-165, Thr-167, Glu-168, Trp-171,
Glu-250, Arg-260, Leu-264, and Ala-267). C, P. eryngii VPL crystal structure (Protein Data Bank code 2BOQ) solvent access surface (showing Ser-158, Val-160,
Glu-161, Trp-164, Glu-243, Lys-253, Arg-257, and Ala-260). D, P. chrysosporium LiPH8 crystal structure (Protein Data Bank code 1LGA) solvent access surface
(showing Asp-165, Leu-167, Glu-168, Trp-171, Lys-260, Asp-264, and Phe-267). Semitransparent solvent access surfaces colored by electrostatic potential (red,
acidic residues; blue, basic residues) are shown. Amino acids are depicted as white or yellow (catalytic tryptophan) van der Waals spheres. The C. subvermispora
peroxidase homology models correspond to those shown in Fig. 1, A and B. The amino acid numbering refers to putative mature sequences after manual
curation of their signal peptide sequences.
ous class II peroxidases, compared with the other basidiomy-
In addition and most interesting is the presence in the C.
cete genomes available. Interestingly, all the class II peroxidase subvermispora genome of two LiP/VP-type genes that had
genes in the C. subvermispora genome appear to be functional, never been detected in this fungus at the gene or protein level
because transcripts for all have been detected in fungal cultures despite intensive efforts in past work. Our initial structural-
(transcriptomic data recently available under GEO accession functional classification of these peroxidases was based on
GSE34636). This class II expansion is particularly true for the whether they contain a conserved exposed tryptophan residue
MnP models, which include a total of 13 genes, a greater num- that could participate in electron transfer reactions or contain
ber than in any sequenced basidiomycete genome except that of formal oxidation sites for Mn2ϩ (61). This analysis suggested
Fomitiporia mediterranea (58).
that one gene encoded a VP (gene 99382) and the other encoded
These C. subvermispora MnPs can be classified as short or an LiP (gene 118677). However, heterologous expression
long MnPs based on structural and catalytic properties. The showed that neither oxidized Mn2ϩ, although both oxidized
latter corresponds to typical MnPs, first described in P. chrys- veratryl alcohol, and therefore we classify both as LiPs.
osporium, and the former appears similar to some P. radiata
Nevertheless, the relatively low catalytic efficiencies of the C.
MnPs that are able to oxidize certain phenols and dyes directly subvermispora LiPs on veratryl alcohol and 1,4-dimethoxyben-
without the involvement of Mn2ϩ (59). The long C. subvermis- zene, as compared with P. chrysosporium LiP, are reminiscent
pora MnPs include five extra long MnPs similar to a thermo- of a typical Pleurotus VP. This property could be related to the
stable MnP found in D. squalens (60), but their catalytic prop- environment near the catalytic tryptophan (Fig. 8), which in the
erties remain to be defined.
two C. subvermispora peroxidases (118677 and 99382) is closer
FIGURE 7. Dendrogram of 376 sequences of basidiomycete heme peroxidases (from GenBankTM and different genomes) showing the position of 25
sequences from the C. subvermispora genome (underlined). The evolutionary analysis was performed with MEGA5 (34) using Poisson distances and
unweighted pair group method with arithmetic mean clustering. Those clusters where C. subvermispora sequences are not included were collapsed, and
prokaryotic origin class I heme peroxidases, including the putative cytochrome c peroxidase from C. subvermispora genome (model 83438), are not included
for simplicity. The three main peroxidase groups (class II in the plant, fungal, and prokaryotic peroxidase superfamily; dye-decolorizing peroxidase (DyP)
superfamily; and HTP superfamily) and the number of sequences in each of them (parentheses) are indicated (right), together with their eight subclusters (A–H),
and the scaffold and model numbers of the different C. subvermispora gene models (parentheses). Peroxidase (see supplemental Table S1 for details) include
five MnP-long enzymes (*) that could be classified as extra long MnPs (60) and two LiP-type enzymes (**) representing LiP/VP transition stages. Additionally,
MnP-atypical corresponds to a MnP type with only two acidic residues at the oxidation site (as found in the Stereum hirsutum genome), TC-LiP corresponds to
the unique LiP from Trametes cervina (46), and GP corresponds to generic peroxidases. See Ruiz-Dueñas and Martínez (29) for references of class II basidiomy-
cete peroxidases (from GenBankTM and genomes).
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to that of P. eryngii VPL than to P. chrysosporium LiPH8 in tion of why veratryl alcohol, a natural metabolite of P. chrys-
osporium, has not so far been found in C. subvermispora.
Alternatively, because veratryl alcohol has been shown to
stabilize LiPs against oxidative inactivation by H2O2 (68), and
assays using synthetic lignins employ large quantities of this
oxidant, the veratryl alcohol requirement may be an artifact of
in vitro reaction conditions. If this second possibility, rather
than the mediator hypothesis, proves to be correct, LiPs prob-
ably cleave lignin by making direct contact with it. Previous
work showing that LiP can bind to lignin favors this second view
(69). Moreover, our observation that the C. subvermispora LiPs
are as effective as P. chrysosporium LiPH8 at oxidizing the bulky
substrate ferrocytochrome c (46, 70) is consistent with our find-
ing that these enzymes all exhibit similar activities on synthetic
lignin.
terms of electrostatic charge distribution. A typical LiP, with
four acidic residues, provides a significantly more electronega-
tive environment than do the three other peroxidases with only
two acidic residues.
Likewise, the ability of the C. subvermispora LiPs to directly
oxidize the dye Reactive Black 5 is a typical property of VPs but
not of LiPs. This property may be due to the absence in the C.
subvermispora peroxidases of bulky residues near the catalytic
tryptophan that could interfere with the binding of Reactive
Black 5; LiPH8 has Phe-267 near Trp-171, whereas the two C.
subvermispora LiPs and P. eryngii VPL have much smaller ala-
nines at analogous positions. The differences in catalytic tryp-
tophan environments of typical VPs and LiPs, as well as their
possible implications in catalysis, have already been discussed
(62).
Acknowledgments—We thank Dr. Daniel Cullen (United States
Department of Agriculture, Forest Products Laboratory, Madison,
WI) and Dr. Rafael Vicuña (Pontificia Universidad Católica de Chile,
Santiago, Chile) for the invitation to participate in the C. subvermis-
pora genome sequencing project. We thank Michael D. Mozuch for
technical assistance with the DHP depolymerization reactions.
Sequencing of the C. subvermispora genome by the Joint Genome
Institute was supported by the Office of Science of the United States
Department of Energy under Contract DE-AC02-05CH11231.
The above catalytic properties, together with sequence-
based phylogenetic analyses that group the C. subvermispora
LiPs with VPs and short MnPs from other fungi, suggest that
the two new C. subvermispora peroxidases represent VP-LiP
transition stages. The discrepancy between the initial structur-
al-functional classification of the 99382 peroxidase (as a VP-
type enzyme) and the lack of Mn2ϩ oxidation activity after het-
erologous expression can be explained by conservation, near its
hypothetical Mn2ϩ oxidation site, of a glutamate residue pres-
ent in LiPs, which could disturb productive Mn2ϩ oxidation
due to an excess of negative charge in this region of the protein.
The Mn2ϩ oxidation activity of our 99382 E183K variant con-
firms this hypothesis.
Note added in proof—While this article was being published, a
description of the whole C. subvermispora genome was published
online (71).
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