Therefore, using conditions of culture identical to those described in [15], our objectives in the present work were: a to produce enough material to purify and characterize this new iso
Trang 1Characterization of a low redox potential laccase
from the basidiomycete C30
Agnieszka Klonowska1, Christian Gaudin2,*, Andre´ Fournel3, Marcel Asso3, Jean Le Petit2,
Michel Giorgi1and Thierry Tron1
1
Laboratoire de Bioinorganique Structurale CNRS UMR 6517 and2Laboratoire d’Ecologie Microbienne, CNRS UMR 6116, Faculte´ desSciencesde St Je´roˆme, Marseille, France;3Laboratoire de Bioe´nerge´tique et Bioinge´nierie desProte´ines,
CNRS UPR9036, Marseille, France
A new exocellular laccase was purified from the
basidio-mycete C30 LAC2 is an acidic protein (pI¼ 3.2)
prefer-entially produced upon a combined induction by copper and
p-hydroxybenzoate The spectroscopic signature (UV/visible
and EPR) of this isoform is typical of multicopper oxidases,
but its enzymatic and physico-chemical properties proved to
be markedly different from those of LAC1, the constitutive
laccase previously purified from the same organism In
particular, the LAC2 kcatvalues observed for the oxidation
of the substrates syringaldazine (kcat ¼ 65 600 min)1),
ABTS (2,2-azino-bis-[3-ethylthiazoline-6-sulfonate] (kcat¼
41 000 min)1) and guaiacol (kcat¼ 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 Polyporusciliatus (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-tion [5,6], among many others [1]
C30 is a white-rot basidiomycete that colonizes the
evergreen oak (Quercusilex L.) leaf litter in the
Mediterra-nean area [7] This fungus has previously been described as
Marasmius quercophilus [7,8], but recent phenotypic and
molecular evidence suggests that it may belong to another
taxon [9,10] Considering the biotope from which C30 was
isolated, delignification is probably essential to this fungus
and laccases may be key enzymes involved in this process [7]
Evidence for the production of at least three different laccases by C30 has previously been obtained [11] The major one, LAC1, was purified, characterized and the correspond-ing gene sequenced [12] However, under the conditions
of culture used, the fungus did not produce other forms in sufficient quantities to allow their full characterization Laccases are generally encoded by gene families [13,14] Expression of these genes can be either constitutive or sensitive to the presence of inducers Consequently, in different fungi, the production of minor laccase forms can
be enhanced under appropriate culture conditions This is the case in C3D for which addition of both copper and p-hydroxybenzoate to the culture broth greatly stimulated the production of three enzyme forms in addition to LAC1 [15] Under these conditions, the fungus was able to syn-thesize the most active of these isoforms at a level similar
to that of LAC1, the constitutive enzyme [15] Therefore, using conditions of culture identical to those described in [15], our objectives in the present work were: (a) to produce enough material to purify and characterize this new isoform named LAC2; (b) to clone the corresponding 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
M A T E R I A L S A N D M E T H O D S
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´roˆme, 13397 Marseille cedex 20, France.
Tel.: 33 491 282856, Fax: + 33 491 983208,
E-mail: thierry.tron@univ.u-3mrs.fr
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 http://www.lbs.fst.u-3mrs.fr
(Received 25 June 2002, revised 4 October 2002,
accepted 22 October 2002)
Trang 2conical flasks containing 600 mL of malt extract/Tween 80
containing 5 mgÆL)1 of CuSO4 and 250 mgÆL)1 of
p-hydroxybenzoate as inducers (malt extract/Tween 80/
Cu/p-hydroxybenzoate) [15] The fungus was cultivated at
30C on a reciprocal shaker (50 r.p.m.) for 5 days [15]
Laccase activity
The routine assay for laccase was based on syringaldazine
oxidation in 0.1M phosphate buffer (pH 5.7) at 30C
2,2¢-Azinobis (3-ethylbenzothiazoline-6-sulfonate) (ABTS)
and guaiacol were also used as substrates under the same
conditions Oxidation of syringaldazine, ABTS and
guaia-col was monitored spectroscopically by absorbance
meas-urements at 525 (e¼ 6.5 · 104
M )1Æcm)1), 420 (e¼ 3.6 ·
104M )1Æcm)1) and 418 nm (e¼ 1.6 · 103
M )1Æcm)1), respectively One unit of laccase oxidizes 1 lmol of substrate
per min
Kinetic parameters (kcatand Km) 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 mM 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
with a step gradient of NaCl: 0.1M, 0.15M, 0.2M, 0.25M,
0.3M, 0.5Mand 1Meach for 25 min More than 50% of
the total laccase activity eluted during the 0.3Mstep The 0.3
MNaCl active fractions were then concentrated and loaded
on a Sephacryl HR 100 column (1.2· 80 cm, Amersham
Pharmacia Biotech Europe GmbH, Freiburg, Germany)
equilibrated with 20 mM phosphate buffer pH 6.0, 0.3M
NaCl and eluted at a flow rate of 0.5 mLÆmin)1
Subse-quently, fractions containing laccase activity were pooled,
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 25C in 0.2M 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
Snapscan 1236 piloted withFOTOLOOK 2.09.6 Legends
were added withCANVAS 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 mMphosphate buffer, pH 6.0 The optimum pH for the enzyme was determined using 0.1M phosphate buffer (pH 5.5–7.5), 0.1M glycine/HCl solution (pH 3.0– 5.0) or 50 mMacetate buffer (pH 4.0–5.5) Its temperature tolerance was determined within the range of 30–85C using phosphate buffer The redox potential (E) of the T1 copper of the two laccases was measured by anaerobic spectrophotometric titration For both LAC1 and LAC2, 50–80 lMof enzyme in 0.1Mphosphate buffer pH 5.7 were titrated after adding 5–10 lM of the following mediators: ferrocenecarboxylic acid, ferrocenedicarboxylic acid and ferroin (Fluka) K2IrCl6 and sodium dithionite were, respectively, used as oxidant and reductant
mRNA isolation and cDNA cloning The fungus was cultivated with inducers as described in [15] Total RNA was extracted from frozen mycelium as des-cribed in [17] Poly(A)+ 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 condiconstruc-tions described in the Marathon cDNA construction kit manual (Clontech) Specific cDNAs were amplified at annealing temperature of 54C, 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 Iused 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
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 3D models for LAC1 and LAC2 were built on the basis of the reported crystal structures of Coprinuscinereuslaccase (GenBank accession no 1A65), using theMODELLERv4.0 [19] Sequence identity with the templates was 57% for LAC1 and 53% for LAC2 Sequences were first aligned withCLUSTALW [20] Then, the alignments were manually adjusted in order to minimize the impact of insertions/ deletions in the final calculations Geometric parameters for the copper coordinations were constrained according to those of C cinereus laccase The top set of calculated structures were finally analyzed with the v3.5 [21]
Trang 3and models visualized with the SWISS-PDBVIEWER v3.7b2
[22]
R E S U L T S
Induction of laccase production
When C30 was grown in shaken liquid culture amended
with both copper sulfate and p-hydroxybenzoic acid, total
laccase activity reached a value of 3.03 UÆmL)1 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 (kmax¼
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 The progressive reduction of the fully oxidized T1 copper was followed spectroscopically by the disappearance of absorption at 608 nm Under our experimental conditions, the E values determined for LAC1 and LAC2 were, respectively, 0.73 and 0.56 V vs normal hydrogen electrode (Table 2) These values are consistent with those previously reported for fungal laccases
Physico-chemical properties and kinetic parameters When LAC2 syringaldazine oxidation was monitored as a function of pH (Table 2), the highest rates were obtained between pH 5.5 and 6, an optimum pH zone 1 unit higher than that previously determined for LAC1 [12] The thermal dependence of this reaction was therefore subsequently tested in phosphate buffer pH 5.7 and maximal syringald-azine oxidation rate was reached at a temperature around
55C (Table 2) This temperature is identical to that found
to be optimum for LAC1 when syringaldazine oxidation was tested at pH 4.5–5
Oxidation of syringaldazine, ABTS and guaiacol were monitored spectroscopically under different pH conditions for LAC1 and LAC2 Kinetic parameters, extracted from Lineweaver–Burk plots, are reported in Table 3 LAC2 exhibited much greater apparent kcatthan those observed for LAC1 but with a lower affinity for all substrates tested The catalytic efficiency (kcat/Km) of LAC2 proved
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 kcatvalues decreased by factor 3, the ratio (kcat/Km) 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 30C, the observed I50 for LAC2 (18 ± 5 lM) is approximately 10 times higher than that for LAC1 (1.5 ± 0.2 lM)
N-Terminal and CNBr peptides sequence analysis Twenty lg of the purified protein were first reduced, then carboxymethylated and subjected to Edman degradation
Table 1 Purification of LAC2 from C30 Activities were measured
using SGZ as substrate.
Step
Protein (mg)
Total activity (U)
Sp act (U/mg)
Yield (%)
Purification (fold) Supernatant 280 10314 37 100 1.0
Ultrafiltration 98 8505 87 83 2.4
DEAE-Sepharose 9 5580 642 54 17.5
Sephacryl S-100 5.5 5135 934 50 25.4
Fig 1 Electrophoresis of purified LAC2 (A) Silver staining of a SDS/
7.5% PAGE; lanes 1 and 5: molecular mass standards; lane 2: 0.5 lg of
purified LAC1; lane 3: 0.5 lg of purified LAC2; lane 4: 10 lg of total
extra cellular proteins (B) Native PAGE stained with p-phenylene
diamine; lane 1: total extra cellular proteins; lane 2: Lac1; lane 3, Lac2;
proteins corresponding to 0.005 U were deposited per lane Dried gels
were scanned with an Agfa Snapscan 1236 using FOTOLOOK 2.09.6.
Legends were added with 7.0.
Trang 4The 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), Trametesvillosa (accession no
L49376 and L78077) and Trametesversicolor (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: Polyporusciliatus
Lcc3-2 (AF176321-1, identity/homology: 81/95%),
Tra-metesversicolor LAC4 (Q12719, identity/homology: 71/
92%), TrametespubescensLAC1A (AF414808-1, identity/
homology: 70/91%) and Trametesvillosa LAC5 (Q99056,
identity/homology: 69/91)
LAC1 and LAC2 homology models For each model, 10 calculated structures were fitted on the reference structure, the C cinereus laccase [23] The general organization of the two models was found to be very close
to that of the reference structure The copper coordination ligands are identical to those present in the reference and the geometry of the T1 copper is basically preserved However, significant differences in folding were found in three regions all located within 10 A˚ around the T1 copper All of these differences are related to gaps present in the initial sequence alignment and corresponding to either insertion or deletion
of one to four residues in the proteins considered (data not shown) 3D representations of region the closest to the T1 copper are given in Fig 2 Polypeptide chain length variations of two residues for LAC1 (Fig 2A) and three residues for LAC2 (Fig 2B) in the loop containing the T1 copper proximal ligand (H396 in C cinereus) induce noticeable structural changes around the copper
D I S C U S S I O N
The basidiomycete C30 secretes at least four different laccases, the proportion of which depends on culture conditions [12,15] We have previously purified and
Fig 2 Comparison of the backbone superposition at the T1 copper site
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)1Æl M )1 ) SGZ LAC1 5.0 1800 1.8 1000
5.7 600 0.9 670 LAC2 5.7 65 600 6.8 9650
GUA LAC1 5.0 ND ND –
5.7 2300 71 30 LAC2 5.7 75 680 1006 76
ABTS LAC1 5.0 3350 10.7 310
5.7 610 2.9 210 LAC2 5.7 41 000 536 80
Table 2 Physico-chemical and EPR parametersof copper sitesof laccases1 and 2 from C30 A // values are in 10)4cm)1units; ND, not determined.
EPR parameters T1 copper T2 copper Enzymes opt pHa T (C) b
E (V) A // g // g ^ A // g // 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.
Trang 5characterized 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
pH for syringaldazine oxidation is close to 4.5–5 for LAC1
and 5.5–6 for LAC2, values that, unlike the data obtained
for several laccases [24], do not correlate with their pI On
the other hand, when tested at their respective optimum pH
values, the two isoforms are the most active at the same
temperature of 55C The amino acid sequences deduced
from lac1, the gene encoding LAC1, and clac2, the cDNA
encoding LAC2, are 69% identical and the two proteins,
LAC1 and LAC2, present very similar optical and EPR
spectra Generally speaking, all the above features of C30
enzymes are similar to those of other white-rot fungi This is
expected as laccases form a group of highly homologous
proteins However, several major physico-chemical features
distinguish LAC1 from LAC2 One is the 170 mV
differ-ence observed between their respective T1 redox
poten-tial measured at pH 5.7 Indeed, with a potenpoten-tial
E ¼ 0.73 V, LAC1 belongs to the group of high redox
potential laccases (T versicolor, T villosa, Rhizoctonia
sol-ani, PleurotusostreatusPOXC and POXA1b, Rigidosporus
lignosus B and D) whereas LAC2, with a potential
E ¼ 0.56 V, belongs to the group of low redox potential
laccases (C cinereus, Myceliophthora thermophila,
Scytali-dium thermophilum) [24–27] To the best of our knowledge,
this difference in potential is the largest so far reported
between two laccases purified from the same organism
Moreover, even though the T1 potentials of R lignosus
laccases B and D and P ostreatus POXC and POXA1b are,
respectively, 40 and 90 mV different [24], all these four
enzymes apparently belong to the high redox potential
group C30 is therefore the first organism for which a
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 Lfi 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 may not be enough to design new targets for mutagenesis
On the other hand, models of our enzymes show substantial structural variations close to the metal center (Fig 2) in a loop where main differences are found when 3D structures, including the recently published structures
of T versicolor [30,31], and Melanocarpusalbomyces[32] laccases are compared (not shown) As folding of the backbone around the metal center was already proposed
to be a major factor affecting the potential in iron–sulfur proteins [29], it seems to us that it would make sense to make mutants in this region
A second distinction between C30 LAC1 and LAC2 enzymes can clearly be made on the basis of their kinetic parameters for the oxidation of phenolic (syringaldazine and guaiacol) as well as nonphenolic (ABTS) substrates (Table 2) Depending on the pH conditions used, we found that LAC2 kcatvalues are one to two orders of magnitude higher than those of LAC1 whereas, the affinity for the three substrates of the former enzyme is lower as reflected by higher Km values Strong differences in Km values are commonly observed for laccases As discussed in previous studies [24,25], the differences in kinetics observed may be the consequence of the variability of certain of the amino acids involved in the substrates channel in specific 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 (kcat/Km) 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 ratio of 10 probably reflects a significant variation in the
Trang 6size of the channel from LAC1 to LAC2 On the other
hand, in a recent study on laccases reactivity with dioxygen
[34], it is speculated from steady state analysis that laccases
have a conserved O2binding domain and that the rate of O2
reduction is dependent on that of substrate oxidation In
our case, this means that LAC2 should reduce dioxygen
much faster than LAC1 and a similar investigation on
oxygen reduction rates must be undertaken on C30 laccases
to further the description of their reactivity
Generally speaking, when compared to kinetic data on
laccases from other fungi, it appears that while LAC1
activity is similar to other laccases for both phenolic and
nonphenolic substrates, LAC2 is a remarkably efficient
enzyme at least on the three substrates tested A comparison
of enzyme efficiency restricted to the group of low redox
potential laccases reveals that, depending on the substrate,
LAC2 appears to be 2–100 times more efficient than its
C cinereus, M thermophila and S thermophilum
counter-parts [24,33] Again, as for their differences in T1 potential,
this is the first report on enzymes produced by a single
organism with such markedly different catalytic efficiency
From a physiological point of view, the constitutive LAC1,
abundant in the culture supernatant [12], exhibits a
relat-ively high affinity for phenols but a relatrelat-ively low capacity
for oxidation when compared to the inducible LAC2 The
consequences of these differences is not yet known but, by
analogy with permeation systems [34] for which such a
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
A C K N O W L E D G E M E N T S
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´glier, Jalila Simaan and Marjorie Sweetko for their critical reading of
the manuscript.
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