Unlike peroxidases, laccases do not require H 2 O 2 or any extra coenzyme for
their reaction but oxygen, and instead of the heme prosthetic group, (at least) four
copper atoms in the active site play key roles in the catalytic cycle [52, 56] (Fig. 3).
The copper atoms are classified based on their spectroscopic features in which the
type-1 copper (T1Cu) has a strong absorption at around 600 nm wavelength in the
oxidized state, which accounts for the blue color of the purified enzymes. While the
type-2 cooper (T2Cu) does not show substantial absorbance feature, the binuclear
type-3 (T3Cu) copper sites exhibit a strong absorbance at 330 nm [52, 56]. The T2
copper and the binuclear T3 coppers form a tri-nuclear copper center (TNC) that
locates around 13 Å apart from the T1 site. While the T1Cu is coordinated by two
His, one Cys, and in many cases, another residue in the axial position (Met in
bacterial/plant laccases or Phe/Leu in fungal laccases), the TNC coppers are
coordinated by eight His residues, two for the T2Cu and three for each of the T3Cu
[52, 56, 57]. The catalytic cycle of a laccase starts with the substrate oxidation
taking place at the T1 site where four electrons of the corresponding reductant are
donated to the resting oxidized (RO) form of the enzyme. To reduce all four Cu(II)
atoms to Cu(I), the electrons first enter the T1 site and are further transferred to the
TNC through the Cys-His pathway. A subsequent peroxide intermediate (PI) is then
formed after the fully reduced TNC reacts with dioxygen followed by a decay of the
PI to a native intermediate (NI) through cleavage of the O–O bond. The cycle is
completed when the NI is slowly decaying to the RO form and with a H 2 O
molecule being released [52, 56].
Most of the fungal and bacterial laccases share the common structural feature of
a globular, three b-barrel cupredoxin-like domain in which the T1Cu site is positioned in domain 1 and the TNC is located between domains 1 and 3. A small
two-domain laccase from Streptomyces coelicolor, named SLAC, was first
described by Machczynski et al. [58] whose structure was elucidated and further
characterized by Skálová et al. [59] (PDB ID: 3CG8). SLAC's structure is composed of six domains that are arranged in the trimeric form of two-domain units.
The T1Cu, in this case, is situated in domain 2, and all copper atoms of TNC are at
the interface of domains 1 and 2. LccED, Laccase, and Multicopper Oxidase
Engineering Database, has assembled the information about sequences and structures of SLAC-like laccases under the homologous family Hfam39 [31]. Many of
the SLAC-like laccases have been found in Actinobacteria, which makes this
phylum an interesting target for mining of new laccases for lignin degradation
[27–29].
Challenges in using laccases for lignin depolymerization include low redox
potentials, especially in many bacterial laccases, and the optimal setup of
laccase-mediator systems (LMS). One approach to increase the redox potential of
bacterial laccases is to exchange the weakly coordinating axial Met residue at the
T1Cu site with non-coordinating Leu or Phe, which are more commonly found in
fungal laccases [60]. One of the most studied bacterial laccases is CotA from
Bacillus spp. which in nature is involved in the formation of pigment in the
endospore's coat. The enzyme has been found to be highly thermostable and to have
a broad substrate scope. Durao et al. [61] observed an increase of approximately
Recent Advances in Enzymatic Conversion of Lignin …
453
their reaction but oxygen, and instead of the heme prosthetic group, (at least) four
copper atoms in the active site play key roles in the catalytic cycle [52, 56] (Fig. 3).
The copper atoms are classified based on their spectroscopic features in which the
type-1 copper (T1Cu) has a strong absorption at around 600 nm wavelength in the
oxidized state, which accounts for the blue color of the purified enzymes. While the
type-2 cooper (T2Cu) does not show substantial absorbance feature, the binuclear
type-3 (T3Cu) copper sites exhibit a strong absorbance at 330 nm [52, 56]. The T2
copper and the binuclear T3 coppers form a tri-nuclear copper center (TNC) that
locates around 13 Å apart from the T1 site. While the T1Cu is coordinated by two
His, one Cys, and in many cases, another residue in the axial position (Met in
bacterial/plant laccases or Phe/Leu in fungal laccases), the TNC coppers are
coordinated by eight His residues, two for the T2Cu and three for each of the T3Cu
[52, 56, 57]. The catalytic cycle of a laccase starts with the substrate oxidation
taking place at the T1 site where four electrons of the corresponding reductant are
donated to the resting oxidized (RO) form of the enzyme. To reduce all four Cu(II)
atoms to Cu(I), the electrons first enter the T1 site and are further transferred to the
TNC through the Cys-His pathway. A subsequent peroxide intermediate (PI) is then
formed after the fully reduced TNC reacts with dioxygen followed by a decay of the
PI to a native intermediate (NI) through cleavage of the O–O bond. The cycle is
completed when the NI is slowly decaying to the RO form and with a H 2 O
molecule being released [52, 56].
Most of the fungal and bacterial laccases share the common structural feature of
a globular, three b-barrel cupredoxin-like domain in which the T1Cu site is positioned in domain 1 and the TNC is located between domains 1 and 3. A small
two-domain laccase from Streptomyces coelicolor, named SLAC, was first
described by Machczynski et al. [58] whose structure was elucidated and further
characterized by Skálová et al. [59] (PDB ID: 3CG8). SLAC's structure is composed of six domains that are arranged in the trimeric form of two-domain units.
The T1Cu, in this case, is situated in domain 2, and all copper atoms of TNC are at
the interface of domains 1 and 2. LccED, Laccase, and Multicopper Oxidase
Engineering Database, has assembled the information about sequences and structures of SLAC-like laccases under the homologous family Hfam39 [31]. Many of
the SLAC-like laccases have been found in Actinobacteria, which makes this
phylum an interesting target for mining of new laccases for lignin degradation
[27–29].
Challenges in using laccases for lignin depolymerization include low redox
potentials, especially in many bacterial laccases, and the optimal setup of
laccase-mediator systems (LMS). One approach to increase the redox potential of
bacterial laccases is to exchange the weakly coordinating axial Met residue at the
T1Cu site with non-coordinating Leu or Phe, which are more commonly found in
fungal laccases [60]. One of the most studied bacterial laccases is CotA from
Bacillus spp. which in nature is involved in the formation of pigment in the
endospore's coat. The enzyme has been found to be highly thermostable and to have
a broad substrate scope. Durao et al. [61] observed an increase of approximately
Recent Advances in Enzymatic Conversion of Lignin …
453