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Kumar 2003
Kumar 2003
DOI: 10.1002/bit.10681
Abstract: Plant and fungal laccases belong to the family nature sequence; multiple sequence alignment; structure
of multi-copper oxidases and show much broader sub- alignment; gene duplication
strate specificity than other members of the family. Lac-
cases have consequently been of interest for potential
industrial applications. We have analyzed the essential
INTRODUCTION
sequence features of fungal laccases based on multiple
sequence alignments of more than 100 laccases. This has
resulted in identification of a set of four ungapped se- Laccases (benzenediol:oxygen oxidoreductase, EC
quence regions, L1–L4, as the overall signature se- 1.10.3.2), a subgroup of the broader class of blue copper
quences that can be used to identify the laccases, distin- oxidases, are widespread in plant and fungal sources. Fun-
guishing them within the broader class of multi-copper gal laccases, as a part of the lignin-degrading enzyme sys-
oxidases. The 12 amino acid residues in the enzymes
serving as the copper ligands are housed within these tem, are present in most of the wood-rotting fungi (Heinzkill
four identified conserved regions, of which L2 and L4 et al., 1998). In addition, the fungal laccases are important
conform to the earlier reported copper signature se- in regular processes like pigment formation (Aramayo et al.,
quences of multi-copper oxidases while L1 and L3 are 1990; Cardenas et al., 2000; Clutterbuck, 1972, 1990), spore
distinctive to the laccases. The mapping of regions L1–L4 formation (Kurtz et al., 1981, 1982), and plant pathogenesis
on to the three-dimensional structure of the Coprinus
cinerius laccase indicates that many of the non-copper-
(Choi et al., 1992; Leatham and Stahmann, 1981). In plants,
ligating residues of the conserved regions could be criti- laccases play a role in lignin polymerization (Ranocha et al.,
cal in maintaining a specific, more or less C-2 symmetric, 1999). Sequence homology analyses suggest that laccases
protein conformational motif characterizing the active could also occur in bacteria such as Mycobacterium tuber-
site apparatus of the enzymes. The observed intraprotein culosis (Alexandre et al., 2000). Laccases have been re-
homologies between L1 and L3 and between L2 and L4 at
both the structure and the sequence levels suggest that
ported from bacteria like Azospirillum lipoferum (Diaman-
the quasi C-2 symmetric active site conformational motif tidis et al., 2000). Among blue copper oxidases, laccases are
may have arisen from a structural duplication event that a subclass of comparatively broader substrate specificity
neither the sequence homology analysis nor the struc- enzymes known to degrade several xenobiotics, i.e., phe-
ture homology analysis alone would have unraveled. Al- nols, anilines, benzene thiols, etc. (Xu, 1996). Conse-
though the sequence and structure homology is not de-
tectable in the rest of the protein, the relative orientation
quently, laccases have evoked particular interest in biotech-
of region L1 with L2 is similar to that of L3 with L4. The nological applications, ranging from biopulping (Wong et
structure duplication of first-shell and second-shell resi- al., 2000) to remediation of wastewater (Novotny et al.,
dues has become cryptic because the intraprotein se- 2000) containing recalcitrant contaminants (D’Annibale et
quence homology noticeable for a given laccase be- al., 1998)
comes significant only after comparing the conservation
pattern in several fungal laccases. The identified motifs, Several plant and fungal laccases are known, having been
L1–L4, can be useful in searching the newly sequenced isolated and characterized spectroscopically and biochemi-
genomes for putative laccase enzymes. © 2003 Wiley Pe- cally. Several other laccases are known based on the protein
riodicals, Inc. Biotechnol Bioeng 83: 386–394, 2003. or cDNA sequence information. Moreover, several putative
Keywords: laccases; multi-copper oxidases; copper sig- plant and fungal laccases have been proposed with the
whole-genome sequencing efforts based on the high homol-
ogy levels with the characterized laccases. Both plant and
Correspondence to: Prof. Pramod Wangikar
Contract grant sponsors: Department of Science and Technology, Gov-
fungal laccases are glycosylated enzymes with the degree of
ernment of India glycosylation varying between 22% and 45%.
Contract grant number: III 5 (57) 99-ET The multi-copper oxidases feature a number of copper
quence variants. The conserved sub-sequences greater than each and every position in all the aligned sequences. The
4 residues in length were examined in greater detail. For sequence logos were constructed with help of the software
each conserved region, a sequence logo was prepared on the Makelogo available online at http://www.bio.cam.ac.uk/cgi-
basis of the frequency of occurrence of various residues at bin/seqlogo/logo.cgi (Schneider et al., 1990).
388 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 83, NO. 4, AUGUST 20, 2003
Table I. Reduction potential and the sub-sequence corresponding to the type I copper signature sequence for various multi-copper oxidases (axial ligand
position (F/L/M) is highlighted in bold).
Reduction
Source Enzyme Partial sequence potential (V) Ref.
Comparison of Signature Sequences at of the alignment analysis are summarized in Figure 2 and
Structural Level Table II. The fungal laccases contain several highly con-
served ungapped regions, distributed almost throughout the
Within the protein, comparisons were performed in order to
length of the proteins (Fig. 2A). The conserved regions
detect any significant repetition of a protein conformational
fold within the overall structure of the C. cinerius laccase common to both plant and fungal laccases are identified in
(PDB code 1A65 and 1HFU). This was achieved using the Figures 2A and B as boxes with the same fill patterns. Some
method of comparison of distance matrices of C␣ atoms regions are distinctive to either the fungal laccases or the
(Peyratout et al., 1994). Initially, an all against all compari- plant laccases (Fig. 2; Table II). The relative positions of the
son of hexapeptide length segments was carried out to ob- conserved regions are identified using the residue number-
tain the root mean square deviation (RMSD) between the C␣ ing of the C. cinerius laccase in case of fungal laccases and
distance matrices. This was done using a program written in the Pinus taeda laccase in case of plant laccases. The po-
C++, that parses a PDB file for C␣ atom coordinates, gen- sitions and lengths of the conserved regions depicted in
erates the hexapeptide distance matrices, and calculates the Figure 2 and Table II did not vary significantly for the
RMSDs. Hexapeptide segments displaying significantly percent consensus criteria between 75% and 85% consen-
low RMSDs were extended on either side to obtain larger sus. Thus, the results are shown for 80% consensus.
and larger regions of conformational homology based on the It is widely accepted that fewer mutations occur in the
maintenance of the RMSD at an acceptably low level. The functionally important residues in an evolutionarily related
statistical significance of the RMSD variation in analyzing family of proteins (Lichtarge et al., 1996). Therefore, to
the extended peptide regions was assessed by standard sta- further analyze the observed sequence conservation pat-
tistical methods. terns, each conserved region greater than seven residues in
length, was converted into a sequence logo, reflecting the
frequency of residue occurrence at each and every position
RESULTS AND DISCUSSION in the sequence. A total of four ungapped conserved regions
Important sequence features of the broader class of multi- are thus detected as the laccase signature sequence com-
copper oxidases, including the residues involved as the cop- posed of the segments L1–L4 starting from the N-terminus.
per ligands, were reported (Solomon et al., 1996), based on The 21-residue L4 region near the C-terminus (residues
a multiple sequence alignment involving two fungal and one 446–466 of C. cinerius laccase) conforms to the earlier
plant laccase, and one sequence each of ascorbate oxidase, reported type I copper signature sequence of multi-copper
Ceruloplasmin, phenoxazinone synthase, bilirubin oxidase, oxidases. This region also conforms to the type II copper
dihydrogeodin oxidase, and FET3 protein. Laccases are signature sequence. Thus, the existing type I or type II cop-
broad substrate specificity enzymes, unlike the rest of the per signature sequences are adequate to describe only a part
multi-copper oxidases, hence there was an interest to exam- of a typical copper-binding region in a laccase or any other
ine if a sequence signature distinctive to the laccase sub- multi-copper oxidase. Notably, the sequence logo for the
group of the multi-copper oxidases could be established. To C-terminus 21-residue L4 region is much more restrictive
this end, 223 plant and fungal laccase sequences or partial and conserved than depicted in the available type I or type
sequences have been collected here from public repositories II copper signature sequences (Table III). A highly homolo-
such as NCBI as well as from patents. After removal of gous L4 sequence also occurs in the plant laccases and is
redundant sequences based on high sequence identity, 64 composed of the residues 535–555 in P. taeda laccase (Fig.
fungal and 40 plant laccase sequences were available for 2B). Comparison of the X-ray crystal structure (Ducros et
present analysis. Multiple sequence alignments were carried al., 1998, 2001) and the sequence logo for the multi-copper
out, separately for the fungal and plant laccases. The results oxidases (Solomon et al., 1996) indicates that three of the
Fungal laccases
64 24 H-W-H-G-(X)9-D-G-(X)5-QCPI
104 21 G-T-(X)-W-Y-H-S-H-(X)3-Q-Y-C-X-D-G-L-X-G-X-(FLIM)-type I
150 4 D-W-Y-H
170 5 L-I-N-G
192 5 G-K-R-Y-R
241 4 Q-R-Y-S
396 8 H-P-X-H-L-H-G-H
446 21 G-X-W-(FIL)-(FLM)-H-C-H-I DAE-X-H-X3-G-(LMF)-X3-(LFM)
Plant laccases
96 24 H-W-H-G-(X)9-D-G-P-(X)3-T-Q-C-P-I
136 8 G-T-L-X-W-H-A-H
208–211 4 T-I-N-G
240–245 6 (VI)-N-A-A-L-N
279–282 4 P-G-Q-T
389–402 4 A-S-X-N-N
436–440 5 F-(ND)-Y-T-G
478–485 8 H-P-X-H-L-H-G-(FYH)
523–534 12 G-W-(X)2-I-R-F-X-A-(DN)-N-P
535–555 21 G-V-W-(FLI)-(FML)-H-C-H-(FMLI)-(DE)-X-H-X2-W-G-L-X-M-X-(WF)
a
The start position of a sub-sequence is shown with reference to the protein sequence of C. cinerius laccase for fungal laccases and P. taeda laccase for
plant laccases.
b
The consensus sequences have been shown as regular expressions [PROSITE pattern (Bucher and Bairoch, 1994; Hoffmann et al., 1999)] and do not
indicate relative frequency of different amino acids at partially conserved positions. For details on protein sequence alignment, refer to legend to Figure 2.
four Cu-ligating residues of the T1 center and two of the residues are highly variable (Fig. 2B). Thus, the 21-residue
Cu-ligating residues of the trinuclear copper cluster (T3 L2 sequence is specific for the fungal laccases, while the L4
copper) (Fig. 2A) are housed within the L4 sequence logo. sequence is common to both the plant and fungal laccases.
In fungal laccases, Phe is the conserved residue at the axial Another large 24-residue conserved segment near the N-
ligand position (residue 17 in the sequence logo), while in terminus characterizes our L1 logo (Fig. 2). This logo, cor-
plant laccases, Leu is the conserved residue at this position. responding to residues 64–87 of C. cinerius laccase, houses
Minor differences between fungal and plant laccase L4 one of the T2 copper ligands and one of the T3 copper
logos occur among the non-ligating residues. ligands. An eight-residue conserved region, residues 396–
In fungal laccases, the 21-residue L2 region near the N- 403 of C. cinerius laccase, houses one ligand each for the
terminus (residues 104–124 of the C. cinerius laccase) also T1, T2, and T3 copper ions (Fig. 2A). This region is con-
conforms to the type I but not to the type II copper signature served in the plant laccases as well (Fig. 2B). In addition to
sequence (Fig. 2A). This region houses two of the Cu- the conserved regions depicted in Figure 2, a few small
ligating residues of the T3 copper center. Only the first eight stretches, 3–7 residues in length, are conserved in the
residues of this region, residues 136–143 of P. taeda lac- aligned laccase sequences. A summary of these sequences is
case, are conserved in the plant laccases while the remaining shown in Table II. When mapped on to the X-ray crystal
Start
Nomenclature positiona Consensus sequence (in PROSITE pattern) Comment
b
Reported copper signature sequences
Type I Undefined G-X-(FYW)-X-(LIVMFYW)-X-(CST)-X8-G-(LM)-X3-(LIVMFYW)
Type II Undefined H-C-H-X3-H-X3-(AG)-(LM)
Observed signature sequences in laccasesb
L1 64 H-W-H-G-X9-D-G-X5-QCPI
L2 104 G-T-X-W-Y-H-S-H-X3-Q-Y-C-X-D-G-L-X-G-X-(FLIM) Conforms to type I signature above
L3 396 H-P-X-H-L-H-G-H
L4 451 G-(PA)-W-X-(LFV)-HCHI-DAE-X-H-X3-G-(LMF)-X3-(LFM) Conforms to type I and II signature above
a
The start position of the signature sequence is shown with respect to C. cinerius laccase.
b
Conserved regions that are at least eight residues in length are shown. Each region houses at least one residue that is ligated with copper.
392 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 83, NO. 4, AUGUST 20, 2003
Figure 3. (A) Peptide backbone of regions 104–124 (L2) and 446–466 (L2) of C. cinerius laccase. (B) Superimposed three-dimensional structure of the
peptide backbone of the regions 104–124 (L2) and 446–466 (L4). The RMSD between the two regions (based on backbone atoms) was found to be 1.15
Å. The ribbon structure was visualized and superimposed using the software MolMol (Koradi et al., 1996). (C) Superimposed three-dimensional structure
of the peptide backbone of the regions 60–124 (shown in purple; comprising L1 and L2) and 396–466 (shown in yellow; comprising L3 and L4); visualized
in WebLab Viewer.
indispensable in both the logos. In fact, a gene duplication Berka RM, Schneider P, Golightly EJ, Brown SH, Madden M, Brown KM,
event has been proposed earlier for the multi-copper oxidase Halkier T, Mondorf K, Xu F. 1997. Characterization of the gene en-
coding an extracellular laccase of Myceliophthora thermophila and
family based on domain recycling, but it has not been sup-
analysis of the recombinant enzyme expressed in Aspergillus oryzae.
ported with sequence conservation pattern. Appl Environ Microbiol 63:3151–3157.
Based on analysis of three-dimensional structure of C.
Bourbonnais R, Paice MG. 1990. Oxidation of non-phenolic substrates. An
cinerius laccase and the multiple sequence analysis of sev- expanded role for laccase in lignin biodegradation. FEBS Lett 267:
eral laccases, we propose that the residues that do not ligate 99–102.
with copper ions were conserved/semi-conserved to main- Bucher P, Bairoch A. 1994. A generalized profile syntax for biomolecular
tain a local three-dimensional fold. In this report, we present sequence motifs and its function in automatic sequence interpretation.
some of the hidden features of laccases, which are not ap- Proc Int Conf Intell Syst Mol Biol 2:53–61.
parent by comparison of the amino acid sequences alone or Cardenas W, Dankert JR. 2000. Cresolase, catecholase and laccase activi-
by comparison of the three-dimensional structures alone. ties in haemocytes of the red swamp crayfish. Fish Shellfish Immunol
The possible gene duplication event is a cryptic one because 10:33–46.
it is unrecognizable at the level of the sequence similarity Choi GH, Larson TG, Nuss DL. 1992. Molecular analysis of the laccase
but is clearly implied by the observed conformation simi- gene from the chestnut blight fungus and selective suppression of its
expression in an isogenic hypovirulent strain. Mol Plant Microb In-
larity shared by the logos. Moreover, from an application
teract 5:119–128.
point of view, the sequence and structure motifs described
Clutterbuck AJ. 1972. Absence of laccase from yellow-spored mutants of
here could be used to search for new laccases and related Aspergillus nidulans. J Gen Microbiol 70:423–435.
enzymes in newly sequenced genomes. Clutterbuck AJ. 1990. The genetics of conidiophore pigmentation in As-
pergillus nidulans. J Gen Microbiol 136:1731–1738.
D’Annibale A, Crestini C, Vinciguerra V, Sermanni GG. 1998. The bio-
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