Characterization of a low redox potential laccase
from the basidiomycete C30
Agnieszka Klonowska
1
, Christian Gaudin
2,
*, Andre
´
Fournel
3
, Marcel Asso
3
, Jean Le Petit
2
,
Michel Giorgi
1
and Thierry Tron
1
1
Laboratoire de Bioinorganique Structurale CNRS UMR 6517 and
2
Laboratoire d’Ecologie Microbienne, CNRS UMR 6116,
Faculte
´
des Sciences de St Je
´
ro
ˆ
me, Marseille, France;
3

)1
) and guaiacol (k
cat
¼ 75 680 min
)1
) are 10–40
times those obtained with LAC1 and the redox potential of
its T1 copper is 0.17 V lower than that of LAC1 (E° ¼
0.73 V). This is the first report on a single organism produ-
cing simultaneously both a high and a low redox potential
laccase. The cDNA, clac2, was cloned and sequenced.
It encodes a protein of 528 amino acids that shares
69% identity (79% similarity) with LAC1 and 81% identity
(95% similarity) with Lcc3-2 from Polyporus ciliatus
(AF176321-1), its nearest neighbor in database. Possible
reasons for why this basidiomycete produces, in vivo,enzyme
forms with such different behaviors are discussed.
Keywords: metalloenzyme; copper; redox potential; laccase;
basidiomycete C30.
Laccases (EC 1.10.3.2) catalyze the oxidation of a large
spectrum of phenolic and nonphenolic substrates with a
concomitant reduction of dioxygen to water [1,2]. They
belong to the blue copper oxidase family and are charac-
terized by the presence of a type 1 copper acting as a primary
electron acceptor from reductant species, and a trinuclear
copper site (one type 2 and two type 3) responsible for the
four electron reduction of dioxygen [3]. These enzymes are
common in plants, fungi, insects and bacteria [1]. In plants,
they may mainly play a role in lignification [4] whereas in
fungi they probably play the opposite role, i.e. delignifica-

coding sequence; and (c) to compare the properties of this
inducible laccase with those of the constitutive LAC1. The
great differences in properties observed between the two
enzymes are discussed in terms of structure–function
relationships.
MATERIALS AND METHODS
Enzyme production
Precultures were carried out on malt extract/Tween 80 as
previously described [12]. They were used to inoculate 3-L
Correspondence to T. Tron, Laboratoire de Bioinorganique
Structurale CNRS UMR 6517, case 432, Faculte
´
des sciences
Saint Je
´
roˆ me, 13397 Marseille cedex 20, France.
Tel.: 33 491 282856, Fax: + 33 491 983208,
E-mail:
Abbreviations: ABTS, 2,2-azino-bis-[3-ethylthiazoline-6-sulfonate].
*Present address: Laboratoire de Bioinorganique Structurale CNRS
UMR 6517.
Note: a web site is available at
(Received 25 June 2002, revised 4 October 2002,
accepted 22 October 2002)
Eur. J. Biochem. 269, 6119–6125 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03324.x
conical flasks containing 600 mL of malt extract/Tween 80
containing 5 mgÆL
)1
of CuSO
4

M
)1
Æcm
)1
),
respectively. One unit of laccase oxidizes 1 lmol of substrate
per min.
Kinetic parameters (k
cat
and K
m
) were estimated using
Lineweaver–Burk plots over a large range of substrate
concentrations.
LAC2 purification
Culture supernatant (3.4 L) was filtered successively
through gauze, paper filter and glass microfiber filters
GFC and GFD (Whatman Ltd, Maidstone, UK), then
concentrated 10-fold by ultrafiltration using YM10 mem-
branes (Amicon, Millipore, Bedford, MA, USA), buffered
with 20 m
M
phosphate, pH 6.0 (buffer A) and finally
applied to an ion-exchange DEAE-Sepharose column
(2.5 · 20 cm, Amersham Pharmacia Biotech Europe
GmbH, Freiburg, Germany) equilibrated with the same
buffer. Proteins were eluted at a flow rate of 4 mLÆmin
)1
.
withastepgradientofNaCl:0.1

concentrated and desalted. Enzyme purity was then con-
firmed by SDS/PAGE.
Enzyme characterization
Determination of protein concentration, syringaldazine
oxidation tests, native and denaturating PAGE and isoelec-
tric focusing analysis were as previously described [12].
Laccase activities were detected by incubating the gels
at 25 °Cin0.2
M
acetate buffer containing 0.2% (w/v)
p-phenylenediamine, at either pH 3.6 or pH 5.2. The
purified protein was subjected to cyanogen bromide treat-
ment as described in [16]; both N-terminal and internal
CNBr peptide sequences were determined by stepwise
Edman degradation. Dried gels were scanned with an Agfa
SnapscanÒ 1236 piloted with
FOTOLOOK
Ò 2.09.6. Legends
were added with
CANVAS
Ò 7.
Laccase absorption was determined on a Uvikon 930
spectrophotometer (Kontron Instruments, Milan, Italy).
X band EPR spectra were recorded on a Bruker (Wissem-
bourg, France) ESP 300 spectrophotometer at 9.3 GHz and
16 K in 20 m
M
phosphate buffer, pH 6.0. The optimum pH
for the enzyme was determined using 0.1
M

+
containing RNAs were purified
with magnetic oligo d(T) beads (PolyA Tract, Promega).
mRNA reverse transcription and cDNA library construc-
tion were performed under the experimental conditions
described in the Marathon cDNA construction kit manual
(Clontech). Specific cDNAs were amplified at annealing
temperature of 54 °C, using a degenerate forward PCR
primer (Eurogentec Seraing, Belgium) AK7 5¢CA(CT)TGG
CA(CT)GGNTT(CT)TT(CT)CA3¢ (identical to the Primer
I used in [18]). This primer is based on the consensus peptide
HWHGFFQ found in copper-binding region I. The univer-
sal Marathon cloning AP1 primer (Clontech) was used as
the reverse primer; nucleotides in parenthese indicate
minimal variations (degeneracy) for the same position.
Two amplicons of 1.9 (cDNA20) and 1.8 kb (cDNA19)
were separated and cloned. After sequencing of their 3¢
ends, specific primers were designed and used to clone full
length cDNAs using the Marathon AP1 primer as forward
primer. Their final sequencing (Genomexpress, Grenoble,
France) confirmed that only cDNA20, amplified with the
reverse primer AK20 5¢CAGAGAACGAACGTA
TGTGCTGG3¢ under the conditions described in the
Marathon cDNa cloning kit manual, encodes the peptides
sequenced from LAC2.
Nucleotide sequence accession no.
The sequence of the C30 laccase cDNA clac2 reported in
this paper has been submitted to GenBank under accession
no. AF491761.
Modeling of LAC1 and LAC2 enzymes

within
5 days. As expected from earlier studies [15], when checked
on native gel electrophoresis, the pattern of laccase isoforms
present in the extracellular fluid showed a high production
of inducible laccases in addition to the constitutive LAC1
(data not shown).
LAC2 purification
Under these culture conditions, the most anionic laccase
isoform is by far the most active [15]. The purification of this
enzyme, named LAC2, was achieved in three steps, allowing
us to recover 5.5 mg of enzyme with a specific activity of
934 UÆmg
)1
, for a final yield of 50% (Table 1). The pure
LAC2 produced a single band both on a SDS/PAGE gel, at
a molecular mass of approximately 65 kDa (Fig. 1A), and
on a native PAGE gel (Fig. 1B).
Spectroscopic characterization
The three types of copper usually present in multicopper
oxidases were detected in purified LAC2. The intense blue
of the enzyme reflected the presence of a T1 copper (k
max
¼
608 nm) whereas that of the binuclear T3 pair was indicated
by a shoulder at 333 nm in the UV/visible spectrum (data
not shown). Values of the constants extracted from the X
band EPR spectrum for the T1 and the T2 coppers
(Table 2) were found to be very similar to those previously
obtained for LAC1 [12].
Redox potential of T1 copper

)ofLAC2proved
to be 10 times that of LAC1 on syringaldazine, whereas
the two laccases exhibited roughly the same efficiency on
guaiacol. With the nonphenolic substrate ABTS, LAC1
efficiency was found to be two to three times that of
LAC2. Although the k
cat
values decreased by factor 3, the
ratio (k
cat
/K
m
) was not much affected when LAC1
kinetics were recorded at pH 5.7 instead of pH 5.0, the
optimum pH for LAC1.
Azide inhibition
Sodium azide inhibition of either syringaldazine or ABTS
oxidation was measured for LAC1 and LAC2. In both
cases, this inhibition was found of noncompetitive type. In
the reaction conditions of optimum pH and at 30 °C, the
observed I
50
for LAC2 (18 ± 5 l
M
) is approximately 10
times higher than that for LAC1 (1.5 ± 0.2 l
M
).
N-Terminal and CNBr peptides sequence analysis
Twenty lg of the purified protein were first reduced, then

Ò 7.0.
Ó FEBS 2002 LAC2 from the basidiomycete C30 (Eur. J. Biochem. 269) 6121
The first 15 residues at the amino terminus are:
AIGPKADLTISNANI. The first six amino acids of this
sequence match perfectly the result obtained on the
laccase contained in fraction D in a preliminary study on
the enhancement of minor laccase production in C30
[15]. We also sequenced a 15-kDa internal peptide and
found that the first 15 residues from this peptide are
AIPNVGTINTDGGVN. A database search showed that
these peptides are closely related to those found in
laccase sequences from basidiomycete CECT 20197
(accession no. U65400), Trametes villosa (accession no.
L49376 and L78077) and Trametes versicolor (accession
no. Y18012).
clac2
cDNA cloning
The cDNA encoding LAC2 was cloned from a PCR
amplified cDNA library. It contains an open reading frame
1584 bp long coding for a protein 528 residue long, a 76-bp
5¢-untranslated region, a 220-bp 3¢-untranslated region and
a 31-bp poylA tail. The amino acid sequence deduced from
the open reading frame contains the peptide sequences
previously characterized from the LAC2 purified protein.
The clac2 ORF is 36 bp longer than the lac1 ORF
(AF162785) and the global identity between the two coding
sequences is 67%. At the protein level, the two enzymes are
69% identical but LAC2 possesses 12 extra amino acids,
seven of which constitute its carboxy terminus. The LAC2
nearest neighbors found in database are: Polyporus ciliatus

of the C. cinereus laccase and the C30 laccase models. The Ca trace of
C. cinereus (1A65) laccase is shown in red. For clarity, only segments
corresponding to loops L333–T341, V387–H399 and H451–A463 and
coordinating residues H396, C452 and H457 are represented. The Ca
traces of 10 calculated models of LAC1 (A) and LAC2 (B) are shown
in grey (coordinating residues, nearly superposable to those of the
C. cinereus laccase, have been omitted for clarity).
Table 3. LAC1 and LAC2 kinetic parameters. ND, not determined.
Substrate Enzymes pH
k
cat
(min
)1
)
K
m
(l
M
)
k
cat
/K
m
(min
)1
Æl
M
)1
)
SGZ LAC1 5.0 1800 1.8 1000

//
g
^
Ref.
LAC1 4.5-5 55 0.73 96 ND ND > 140 ND ND 6
LAC2 5.5-6 55 0.56 88 2.165 2.025 172 2.25 2.027 This work
a
Values obtained with SGZ as substrate.
b
Temperature for which the main activity is reached with SGZ as substrate.
6122 A. Klonowska et al. (Eur. J. Biochem. 269) Ó FEBS 2002
characterized LAC1, the most abundant enzyme produced
by C30 [12]. The purification of a second laccase (LAC2)
from this fungus allows us to compare enzymes, the
synthesis of which is regulated differently. Indeed, LAC1
is produced under all the conditions we have tested so far
and thus is probably a constitutive form. On the other hand,
LAC2, which is almost absent in noninduced cultures,
becomes one of the most prominent laccases secreted when
the growth medium is supplemented with copper and
p-hydroxybenzoate; it can therefore be considered an
inducible enzyme [15]. Such differences in their patterns of
expression suggest a distinct physiological role for these two
isoforms and, although they share basic properties, the large
variation in catalytic activity for both phenolic and dyes
supports this idea.
The C30 laccase isoforms we have detected so far all have
an apparent molecular mass close to 65 kDa and, except for
a still-uncharacterized laccase, are all acidic proteins with pI
ranging from 3.2 (LAC2) to 3.6 (LAC1) [15]. The optimum

simultaneous production of high redox and low redox
potential laccases is reported.
Several attempts have been made to correlate the redox
potential variations found in laccases to the nature of the
specific amino acids present in their active site as their
oxidative capabilities appear tightly linked to this param-
eter [24,28]. The replacement F463M in a T. villosa
(accession no. AAC41686) high redox potential laccase
provides a fourth coordinating axial ligand to the T1
copper resulting in a 100-mV drop of its potential [27]. On
the other hand, the idea that the occurrence of a
phenylalanine residue might correlate with a high redox
potential in laccases was ruled out by the site directed
replacement of L fi F in two laccases [26]. The presence
of an F residue at the corresponding position in the
sequence of both the C30 high redox potential LAC1 and
low redox potential LAC2 sequences support this conclu-
sion. Similarly, the replacement of the LEA amino acid
triplet located immediately after the distal T1 coordinating
histidine (H456 in C. cinereus, accession no. 1A65) in the
high redox potential R. solani laccase (accession no.
Q02081), by a SVG amino acid triplet found in the low
redox potential M. thermophila (accession no. AAE35046)
and vice versa did not affect significantly the E° of the
recombinant enzymes [26]. In our study, the presence of a
LEA tripeptide both in the C30 LAC1 and LAC2
enzymes correlates well with these results. In an effort
to gain new insights into the factors influencing the
potential of the T1 copper in laccases, data on eight high
redox potential and four low redox potential enzymes

A substantial variation in the folding of the T1 copper
pocket of the two enzymes, such as that mentioned above to
explain their difference in T1 potential, could also account
for their specific interaction with the substrates. In laccases,
enzyme efficiency (k
cat
/K
m
) correlates with the redox
potential of the substrates [24] and the two C30 laccases
behave more or less this way. In fact, like already observed
by Garzillo et al. in their study on T. trogii, R. lignosus and
P. ostreatus laccases [25], LAC1 and LAC2 activities on
phenolic compounds seem only partly related to their
specific redox capabilities. Indeed, LAC2 appears to be two
to 10 times more efficient than LAC1 on phenolic substrates
although with a 170-mV lower T1 copper redox potential.
As phenol oxidation involves release of a proton, factors
like hydrogen bonding or the extent of protonation of
ionizable residues in the vicinity of the T1 copper probably
have considerable effects on the overall efficiency.
Differences between LAC1 and LAC2 are not restricted
to the oxidation site as the two enzymes also react differently
toward sodium azide, an inhibitor known to bind to the
oxygen reduction site. It is likely that a channel governs the
accessibility to the T2/T3 cluster. Therefore, a LAC2/LAC1
I
50
ratio of 10 probably reflects a significant variation in the
Ó FEBS 2002 LAC2 from the basidiomycete C30 (Eur. J. Biochem. 269) 6123

contrast between affinity and rate is often observed, we
could interpret the differences in laccase properties as a need
for the organism to maintain both a low capacity/high
specificity system when substrate level is low and a high
capacity/low specificity system when the substrate is abun-
dant.
In conclusion, we have demonstrated that LAC2, a laccase
produced by the basidiomycete C30 following copper and
p-hydroxybenzoate induction, is a low redox potential
enzyme with unusually high oxidative capabilities. The
kinetic data obtained both on phenolic and nonphenolic
substrates indicate that LAC2 might be a good catalyst for
the transformation of different substrates. As laccases are
generally produced as a number of isoenzymes encoded by
multigene families, the expression of which varies from
fungus to fungus, it is highly probable that other fungi
contain the equivalent of LAC2. A search for the appropri-
ate conditions of expression of a given activity being empirical
and time consuming, it will probably be more efficient to use
a heterologous expression system for laccase activities to
find other enzymes with high oxidative capacities.
ACKNOWLEDGEMENTS
A. K. is the recipient of an Agence de l’Environement et de la Maıˆtrise
de l’Energie (ADEME) fellowship. This work was in part supported by
a grant from the Conseil Ge
´
ne
´
ral 13. We thank Gilles Iacazio, Marius
Re

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