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Laccases: Structure, reaction, distribution

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DOI: 10.1016/j.micron.2003.10.029 · Source: PubMed

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 Micron 35 (2004) 93–96
www.elsevier.com/locate/micron

Laccases: structure, reactions, distribution

Harald Claus*
Institut für Molekulare Biophysik, University of Mainz Jakob-Welder-Weg 26, 55128 Mainz, Germany

Abstract
Laccases (EC 1.10.3.2, p-diphenol: dioxygen oxidoreductases) are multi-copper proteins that use molecular oxygen to oxidize various
aromatic and non-aromatic compounds by a radical-catalyzed reaction mechanism. The enzymes are involved in the pathogenicity, immunity
and morphogenesis of organisms and in the metabolic turnover of complex organic substances such as lignin or humic matter. Owing to their
high non-specific oxidation capacity, laccases are useful biocatalysts for diverse biotechnological applications. Until recently, laccases were
only found in eukaryotes (fungi, higher plants, insects), but now there is strong evidence for their widespread distribution in prokaryotes and
the first crystal structure of a bacterial laccase is already available. Phylogenetically, laccases are members of the multi-copper protein family
including ascorbate oxidase, ceruloplasmin and bilirubin oxidase.
q 2003 Elsevier Ltd. All rights reserved.
Keywords: Laccases; Procaryotes; Biocatalysts

1. Structure is methionine in the bacterial (CotA) and leucine or

Fungal laccases often occur as isoenzymes that oligo-
merize to form multimeric complexes. The molecular mass
of the monomer ranges from about 50 to 100 kDa. An
important feature is a covalently linked carbohydrate moiety
(10 – 45%), which may contribute to the high stability of the
enzymes. The crystal structure of the fungal laccase from
Coprinus cinerius laccase is now available (Ducros et al.,
1998, 2001). More recently the spore coat protein cotA of
Bacillus subtilis has been identified as a laccase (Hullo et al.,
2001; Martins et al., 2002) and the crystal structure has been
presented (Enguita et al., 2003).
For the catalytic activity a minimum of four copper
atoms per active protein unit is needed:
Type 1: paramagnetic ‘blue’ copper, absorbance at
610 nm (ox.).
Type 2: paramagnetic ‘non-blue’ copper.
Type 3: diamagnetic spin-coupled copper-copper pair,
absorbance at 330 nm (ox.).
Type 1 copper has a trigonal coordination, with two
histines and a cysteine as conserved equatorial ligands
and one position usually variable. This axial ligand
* Fax: þ49-6131-3923557.
E-mail address: hcdclaus@t-online.de (H. Claus).
phenylalanine in fungal laccases. It has been widely argued
that this axial position ligand strongly influences the
oxidation potential of the enzyme, possibly providing the
mechanism for regulating its activity. A mutation from
phenylalanine to methionine significantly lowered the
oxidation potential of a fungal laccase from Trametes
villosa (Kumar et al., 2003). Type I copper confers the
typical blue colour to multicopper proteins, which results
from the intense electronic absorption caused by the
covalent copper –cysteine bond. Due to its high redox
potential of ca. þ 790 mV, type 1 copper is the site where
substrate oxidation takes place. Type 2 copper shows no
absorption in the visible spectrum and reveals paramagnetic
properties in EPR studies. It is stategically positioned close
to the type 3 copper, a binuclear center spectroscopically
characterized by an electron adsorption at 330 nm (oxidized
form) and by the absence of an EPR signal as the result of
the anti-ferromagnetic coupling of the copper pair. The type
3 copper centre is also the common feature of another
protein superfamily including the tyrosinases and haemo-
cyanins (Decker and Terwilliger, 2000).
Type 2 and type 3 copper form a trinuclear cluster, where
reduction of molecular oxygen and release of water takes
place. Type 2 copper is coordinated by two and type 3
copper atoms by six histidines. The strong anti-ferromag-
netical coupling between the two type 3 copper atoms, is
maintained by a hydroxyl bridge (Fig. 1).

0968-4328/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.micron.2003.10.029
94 H. Claus / Micron 35 (2004) 93–96
Fig. 1. Copper centers of the laccase (CotA) from B. subtilis (adapted from Enguita et al., 2003).
Multiple sequence alignments of more than 100 laccases
resulted in identification of four ungapped sequence
regions, L1 –L4, as the overall signature of laccases,
distinguishing them within the broader class of multi-
copper oxidases (Kumar et al., 2003). The 12 amino acid
residues in the enzymes serving as the copper ligands are
housed within these conserved regions. The amino acid
ligands of the trinuclear cluster are the eight histines,
which occur in a highly conserved pattern of four HXH
motifs. In one of this motifs, X is the cysteine bound to the
T1 copper while each of the histines is bound to one of the
two type 3 coppers. Intraprotein homologies between
signatures L1 and L3 and between L2 and L4 suggest the
occurrence of duplication events.
2. Substrates and reaction mechanisms
The various copper centres of laccases drive electrons
from a reducing substrate to molecular oxygen without
releasing toxic peroxide intermediates. This is accom-
plished by four monoelectronic oxidations of the substrate
catalysed by the type 1 copper. The electrons are further
transferred to the trinuclear cluster, where reduction of
molecular oxygen and release of water takes place. The
oxidation of substrates creates reactive radicals that can
undergo non-enzymatic reactions:
Cross-linking of monomers: The enzymatic oxidation of
phenolic compounds and anilines by laccases generate
radicals that react with each other to form dimers,
oligomers or polymers covalently coupled by C – C, C –O
and C – N bonds. In soils, natural and xenobiotic phenolics
or aromatic amines can thus be bound to the organic humic
matrix. In the case of substituted compounds, the reaction
can be accompanied by partial demethylations and
dehalogenations. This capacity of laccases is the basis for
their potential use to detoxify contaminated soils or waste
waters (Filip and Claus, 1995; Durán and Esposito, 2000).
In higher plants, the cross-linking of phenolic precursors
by laccases is one part in the ligninification process. In
insects, the laccase-catalysed oxidative coupling of cate-
chols with proteins may be involved in cuticle sclerotiza-
tion (Kramer et al., 2001). In microorganisms cross-linking
of proteins residues, eg. tyrosine to dityrosine, has been
discussed as the function of laccases in the assembly of the
heat and UV-resistant Bacillus spores (Hullo et al., 2001;
Martins et al., 2002).
Degradation of polymers: Laccases are involved in the
degradation of complex natural polymers, such as lignin or
humic acids (Claus and Filip, 1998). The reactive radicals
generated, lead to the cleavage of covalent bonds and to the
release of monomers. Because of steric hindrance,
the enzymes might not come directly into contact with the
polymers. Instead small organic compounds or metals that
can also be oxidized and activated by laccases,
e.g. veratrylalcohol, 3-hydroxy-anthranilic acid and
manganese mediate the radical-catalysed depolymerization.
Non-physiological redox-mediators are used in biotechno-
logical processes to increase the oxidation potential of
laccases (Claus et al., 2002).
 H. Claus / Micron 35 (2004) 93–96
Ring cleavage of aromatics: In several cases a laccase-
catalyzed ring-cleavage of aromatic compounds has been
reported. This reaction is of biotechnological interest in
view of the degradation of xenobiotics like nitroaromatics
and synthetic dyes (Durán and Esposito, 2000; Claus et al.,
2002).
3. Distribution in prokaryotes
Until recently laccases were found only in eukaryotes,
e.g. fungi, plants, insects (Mayer and Staples, 2002).
Now there is increasing evidence for the existence
in prokaryotes of proteins with typical features of the
multi-copper oxidase enzyme family (Alexandre and
Zhulin, 2000; Claus, 2003). Corresponding genes have
been found in gram-negative and gram-positive bacteria,
including species living in extreme habitats, e.g. in
Oceanobacillus iheyensis or Aquifex aeolicus and in the
archaebacterium Pyrobaculum aerophilum. Early reports of
laccases in actinomycetes were based on rather non-specific
substrate reactions, but have been verified for Streptomyces
griseus (Freeman et al., 1993), S. griseus (Endo et al., 2002)
and S. lavendulae (Suzuki et al., 2003). The first convincing
data for a prokaryotic laccase activity were presented for
Azospirillum lipoferum (Givaudan et al., 1993). It is a
multimeric enzyme, composed of a catalytic subunit and
one or two larger chains. After SDS-PAGE, three bands are
present with molecular masses of 48.9, 97.8 and 179.3 kDa
(Diamantidis et al., 2000). Marinomonas mediterranea is
a melanogenic marine bacterium expressing both an
SDS-activated tyrosinase and a laccase (Sanchez-Amat
and Solano, 1997). The laccase, which was heterologously
expressed in Escherichia coli, revealed the typical
copper-binding domains of laccases and two additional
potential copper-binding sites near the N-terminus.
The latter may be responsible for the tyrosinase-activity of
the enzyme (Sanchez-Amat et al., 2001). A laccase-like
enzyme activity was found in spores of a Bacillus
sphaericus strain (Claus and Filip, 1997). Recently
the spore protein CotA of Bacillus subtilis has been
recognized to be a laccase (Hullo et al., 2001). Mutants in
the gene encoding CotA lost the ability to produce a
brownish spore pigment. The latter may protect the spores
against stress factors such as UV radiation or
hydrogen peroxide. The protein, which was overexpressed
in E. coli, has a molecular mass of 65 kDa, an isoelectric
point of 7.7 and is highly thermostable (Martins et al.,
2002).
In conclusion, laccases are exceptionally versatile
enzymes, catalyzing one basic reaction from which all its
activities originate (Mayer and Staples, 2002). They are
ubiquitous, being distributed in all domains of life; more
investigations are needed for a better understanding of their
physiological importance and to further define their great
biotechnological potential.
95
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