Combined Sequence and Structure

Analysis of the Fungal Laccase Family

S. V. Suresh Kumar,1,2 Prashant S. Phale,2 S. Durani,2

Pramod P. Wangikar1,2
1
Department of Chemical Engineering, Indian Institute of Technology,
Bombay, Powai Mumbai, 400 076 India; fax: 91-22-2572 6895/2572 3480;
e-mail: pramodw@che.iitb.ac.in
2
BJM School of Biosciences and Bioengineering, Indian Institute of
Technology, Bombay, Powai Mumbai, 400 076 India
Received 23 March 2002; accepted 27 January 2003

DOI: 10.1002/bit.10681

Abstract: Plant and fungal laccases belong to the family
of multi-copper oxidases and show much broader sub-
strate specificity than other members of the family. Lac-
cases have consequently been of interest for potential
industrial applications. We have analyzed the essential
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-
quence regions, L1–L4, as the overall signature se-
quences that can be used to identify the laccases, distin-
guishing them within the broader class of multi-copper
oxidases. The 12 amino acid residues in the enzymes
serving as the copper ligands are housed within these
four identified conserved regions, of which L2 and L4
conform to the earlier reported copper signature se-
quences of multi-copper oxidases while L1 and L3 are
distinctive to the laccases. The mapping of regions L1–L4
on to the three-dimensional structure of the Coprinus
cinerius laccase indicates that many of the non-copper-
ligating residues of the conserved regions could be criti-
cal in maintaining a specific, more or less C-2 symmetric,
protein conformational motif characterizing the active
site apparatus of the enzymes. The observed intraprotein
homologies between L1 and L3 and between L2 and L4 at
both the structure and the sequence levels suggest that
the quasi C-2 symmetric active site conformational motif
may have arisen from a structural duplication event that
neither the sequence homology analysis nor the struc-
ture homology analysis alone would have unraveled. Al-
though the sequence and structure homology is not de-
tectable in the rest of the protein, the relative orientation
of region L1 with L2 is similar to that of L3 with L4. The
structure duplication of first-shell and second-shell resi-
dues has become cryptic because the intraprotein se-
quence homology noticeable for a given laccase be-
comes significant only after comparing the conservation
pattern in several fungal laccases. The identified motifs,
L1–L4, can be useful in searching the newly sequenced
genomes for putative laccase enzymes. © 2003 Wiley Pe-
riodicals, Inc. Biotechnol Bioeng 83: 386–394, 2003.
Keywords: laccases; multi-copper oxidases; copper sig-
Correspondence to: Prof. Pramod Wangikar
Contract grant sponsors: Department of Science and Technology, Gov-
ernment of India
Contract grant number: III 5 (57) 99-ET
nature sequence; multiple sequence alignment; structure
alignment; gene duplication
INTRODUCTION
Laccases (benzenediol:oxygen oxidoreductase, EC
1.10.3.2), a subgroup of the broader class of blue copper
oxidases, are widespread in plant and fungal sources. Fun-
gal laccases, as a part of the lignin-degrading enzyme sys-
tem, are present in most of the wood-rotting fungi (Heinzkill
et al., 1998). In addition, the fungal laccases are important
in regular processes like pigment formation (Aramayo et al.,
1990; Cardenas et al., 2000; Clutterbuck, 1972, 1990), spore
formation (Kurtz et al., 1981, 1982), and plant pathogenesis
(Choi et al., 1992; Leatham and Stahmann, 1981). In plants,
laccases play a role in lignin polymerization (Ranocha et al.,
1999). Sequence homology analyses suggest that laccases
could also occur in bacteria such as Mycobacterium tuber-
culosis (Alexandre et al., 2000). Laccases have been re-
ported from bacteria like Azospirillum lipoferum (Diaman-
tidis et al., 2000). Among blue copper oxidases, laccases are
a subclass of comparatively broader substrate specificity
enzymes known to degrade several xenobiotics, i.e., phe-
nols, anilines, benzene thiols, etc. (Xu, 1996). Conse-
quently, laccases have evoked particular interest in biotech-
nological applications, ranging from biopulping (Wong et
al., 2000) to remediation of wastewater (Novotny et al.,
2000) containing recalcitrant contaminants (D’Annibale et
al., 1998)
Several plant and fungal laccases are known, having been
isolated and characterized spectroscopically and biochemi-
cally. Several other laccases are known based on the protein
or cDNA sequence information. Moreover, several putative
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
fungal laccases are glycosylated enzymes with the degree of
glycosylation varying between 22% and 45%.
The multi-copper oxidases feature a number of copper

© 2003 Wiley Periodicals, Inc.

centers as part of their overall catalytic apparatus. Based on
spectroscopic analysis, which reflects geometric and elec-
tronic features, copper centers are differentiated as the type
1 (T1), or blue copper center, or a trinuclear copper cluster,
which is composed of one type 2 (T2) and two type 3 (T3)
center (Solomon et al., 1996). Despite strong similarity in
their EPR parameters, the reduction potential of the T1 cen-
ter can vary widely in different enzymes, from approxi-
mately 0.465 V in Myceliophthora thermophila laccase to
0.775 V in Polyporus versicolor laccase. Sequence homol-
ogy analysis among multi-copper oxidases has shown that
the conserved residues ligating with Cu at the T1 copper
center are two histidines and a cysteine. The crystal struc-
ture of only one fungal laccase is thus far known (Ducros et
al., 1998, 2001). As shown in Figure 1, the T1 center of this
Coprinus cinerius laccase (Ducros et al., 1998) features
His396, Cys452, and His457 as the copper ligands. In ad-
dition, the T1 center features a non-ligating Leu462 at an
axial ligand position. It has been widely argued that this
axial position ligand strongly influences the reduction po-
tential of an enzyme, possibly providing the mechanism for
tuning of its reactivity (Xu et al., 1999). Most but not all
fungal laccases carry a Leu or Phe at this conserved position
ten residues downstream from the Cys (Table I). Several
other multi-copper oxidases (non-laccases) feature a Met at
this position and display a relatively diminished reduction
potential. A mutation of this residue in Trametes villosa
laccase from Phe to Met was shown to result in significant
lowering of the reduction potential of this fungal enzyme
(Xu et al., 1998, 1999) (Table I). Based on the pattern of
conservation noted for some of the amino acid ligands at the
T1 copper center, a 21-residue signature sequence (Askwith
et al., 1994; Mann et al., 1988; Messerschmidt et al., 1990;
Ouzounis et al., 1991) was defined for the multi-copper
oxidases as G-X-[FYW]-X-[LIVMFYW]-X-[CST]-X8-G-
[LM]-X3-[LIVMVYW]. An X in this signature represents
an undefined residue while the multiple letters within brack-
ets represent a partially conserved residue. Of the ligands of
the T1 center shown in Figure 1, Cys452, His456, and
Leu462 lie within the signature sequence defined above.
The reasons for the fully or partially conserved nature of
rest of the residues within the signature sequence are not
known. The overall function of the T1 center is that of
long-range intramolecular electron transfer, from the T1
center to the trinuclear copper cluster approximately 13 Å
away, along a cysteine–histidine pathway (Fig. 1b). The
amino acid ligands of the trinuclear cluster are the eight
histidines, which occur in a highly conserved pattern of four
HXH motifs in the enzymes. In one of these motifs, X is the
cysteine bound to the T1 copper while each of the histidines
is bound to one of the two T3 coppers. The HXH motifs are
separated from one another by segments of between 25 and
175 residues and are likely to be brought close in a com-
posite catalytic apparatus by an aspect of protein folding.
An additional, 12-residue-long type II signature sequence
(Askwith et al., 1994; Mann et al., 1988; Messerschmidt and
SURESH KUMAR ET AL.: ANALYSIS OF THE FUNGAL LACCASE FAMILY
Huber, 1990; Ouzounis and Sander, 1991) has been defined
as H-C-H-X3-H-X3-[AG]-[LM].
It is possible that laccases, due to their comparatively
broader substrate specificities (Xu, 1996), share a sequence
signature that can distinguish them as a specific subgroup of
(Bourbonnais et al., 1990; Xu et al., 1996) the multi-copper
oxidase family. In this report, we present results of an analy-
sis of over 100 plant and fungal laccases and the resultant
identification of a sequence signature that uniquely charac-
terizes the laccases as a distinctive subgroup of enzymes of
the multi-copper oxidase family. The signature, composed
of four ungapped sequence segments L1–L4 ranging from 8
to 24 residues in length and scattered across almost the
entire length of the protein, contains all the amino acid
ligands of the copper centers and other completely or par-
tially conserved residues that seem critical for the mainte-
nance of a quasi C-2 symmetric protein conformational fold
characterizing the catalytic apparatus in the enzymes, which
may have originated from a gene duplication event. The
possible duplication, of functionally important residues at
structural level, is of a cryptic nature, nondetectable at the
sequence level alone but clearly identifiable by the com-
bined analysis of the intra-protein sequence and conforma-
tion homologies that we have identified in this study.
MATERIALS AND METHODS
The laccase sequences of plant and fungal origin were ob-
tained from the NCBI protein sequence repository (http://
www.ncbi.nlm.nih.gov) as well as from the U.S. Patent Of-
fice web site (http://www.uspto.gov). Some of the se-
quences are for laccases experimentally characterized by
others, while the rest are based on DNA or cDNA sequences
for putative laccases or laccase precursors based on high
sequence homology with the known laccases.
Analysis of Amino Acid Sequences
Of the available 223 laccase sequences, redundant se-
quences were removed to obtain 64 fungal laccases and 40
plant laccases. Two sequences having identity of greater
than 95% were considered redundant, and one of the two
was omitted from the analysis. The final non-redundant se-
quences contain both type I and type II copper signature
sequences (Bucher and Bairoch, 1994; Hofmann et al.,
1999). Multiple sequence alignments were undertaken with
help of the software ClustalW (Thompson et al., 1994) us-
ing the amino acid substitution matrix BLOSUM 62 (Heni-
koff et al., 1992, 1996) the recommended procedure for
regular comparisons of protein sequences. For multiple se-
quence alignment, gap opening penalty of 8 and gap exten-
sion penalty of 2 was used. Phylogenetic trees were con-
structed using the software Phylip 1.5.2 (Page, 1996). From
the results of multiple sequence alignment, the “80% con-
sensus sequence” was obtained by writing the amino acid
that appears at a given position in at least 80% of the se-
387
Figure 1. View of the ligands at T1 copper and trinuclear copper centers in C. cinereus laccase (a fungal laccase; PDB code 1A65) (adapted from Ducros
et al., 1998, 2001). (A) Three-dimensional structure of backbone of entire domain of C. cinereus laccase showing all the copper ions at T1 and T3 copper
centers. (B and C) Amino acid ligands in the vicinity of the T1 copper center. and the trinuclear copper center with T2 and T3 copper. For T1 copper
His396, His457, and Cys452 are equatorial ligands while Leu462 is an axial ligand. The His451–Cys452 electron bridge is highlighted.

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).
Source Enzyme
T. villosa (wild type) Laccase
T. villosa (Phe463Leu) Laccase
T. villosa (Phe463Met) Laccase
P. versicolor Laccase
Rhizoctonia solani Laccase
M. thermophila Laccase
Scytalidium thermophilum Laccase
C. cinereus Laccase
Zucchini Ascorbate oxidase
Human Ceruloplasmin
Myrothecium verrucaria Bilirubin oxidase
Comparison of Signature Sequences at
Structural Level
Within the protein, comparisons were performed in order to
detect any significant repetition of a protein conformational
fold within the overall structure of the C. cinerius laccase
(PDB code 1A65 and 1HFU). This was achieved using the
method of comparison of distance matrices of C␣ atoms
(Peyratout et al., 1994). Initially, an all against all compari-
son of hexapeptide length segments was carried out to ob-
tain the root mean square deviation (RMSD) between the C␣
distance matrices. This was done using a program written in
C++, that parses a PDB file for C␣ atom coordinates, gen-
erates the hexapeptide distance matrices, and calculates the
RMSDs. Hexapeptide segments displaying significantly
low RMSDs were extended on either side to obtain larger
and larger regions of conformational homology based on the
maintenance of the RMSD at an acceptably low level. The
statistical significance of the RMSD variation in analyzing
the extended peptide regions was assessed by standard sta-
tistical methods.
RESULTS AND DISCUSSION
Important sequence features of the broader class of multi-
copper oxidases, including the residues involved as the cop-
per ligands, were reported (Solomon et al., 1996), based on
a multiple sequence alignment involving two fungal and one
plant laccase, and one sequence each of ascorbate oxidase,
Ceruloplasmin, phenoxazinone synthase, bilirubin oxidase,
dihydrogeodin oxidase, and FET3 protein. Laccases are
broad substrate specificity enzymes, unlike the rest of the
multi-copper oxidases, hence there was an interest to exam-
ine if a sequence signature distinctive to the laccase sub-
group of the multi-copper oxidases could be established. To
this end, 223 plant and fungal laccase sequences or partial
sequences have been collected here from public repositories
such as NCBI as well as from patents. After removal of
redundant sequences based on high sequence identity, 64
fungal and 40 plant laccase sequences were available for
present analysis. Multiple sequence alignments were carried
out, separately for the fungal and plant laccases. The results
SURESH KUMAR ET AL.: ANALYSIS OF THE FUNGAL LACCASE FAMILY
Reduction
Partial sequence potential (V) Ref.
452-HCHIDFHLEAGF-463 0.79 Yaver et al., 1996
452-HCHIDFHLEAGL-463 0.74 Xu et al., 1998
452-HCHIDFHLEAGM-463 0.68 Xu et al., 1998
451-HCHIDFHLEAGF-462 0.79 Jonsson, et al., 1995
459-HCHIDWHLEAGL-470 0.71 Wahleithner et al., 1996
502-HCHIAWHVSGGL-513 0.47 Berka et al., 1997
506-HCHIAWHVSGGL-517 0.51 Xu et al., 1995
451-HCHIEFHLMNGL-462 0.55 Schneider et al., 1999
506-HCHIEPHLHMGM-517 0.34
997-HCHVTDHIHAGM-1008 0.49–0.58
495-HCHNLIHEDHDM-506 0.48–0.49
of the alignment analysis are summarized in Figure 2 and
Table II. The fungal laccases contain several highly con-
served ungapped regions, distributed almost throughout the
length of the proteins (Fig. 2A). The conserved regions
common to both plant and fungal laccases are identified in
Figures 2A and B as boxes with the same fill patterns. Some
regions are distinctive to either the fungal laccases or the
plant laccases (Fig. 2; Table II). The relative positions of the
conserved regions are identified using the residue number-
ing of the C. cinerius laccase in case of fungal laccases and
the Pinus taeda laccase in case of plant laccases. The po-
sitions and lengths of the conserved regions depicted in
Figure 2 and Table II did not vary significantly for the
percent consensus criteria between 75% and 85% consen-
sus. Thus, the results are shown for 80% consensus.
It is widely accepted that fewer mutations occur in the
functionally important residues in an evolutionarily related
family of proteins (Lichtarge et al., 1996). Therefore, to
further analyze the observed sequence conservation pat-
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
in the sequence. A total of four ungapped conserved regions
are thus detected as the laccase signature sequence com-
posed of the segments L1–L4 starting from the N-terminus.
The 21-residue L4 region near the C-terminus (residues
446–466 of C. cinerius laccase) conforms to the earlier
reported type I copper signature sequence of multi-copper
oxidases. This region also conforms to the type II copper
signature sequence. Thus, the existing type I or type II cop-
per signature sequences are adequate to describe only a part
of a typical copper-binding region in a laccase or any other
multi-copper oxidase. Notably, the sequence logo for the
C-terminus 21-residue L4 region is much more restrictive
and conserved than depicted in the available type I or type
II copper signature sequences (Table III). A highly homolo-
gous L4 sequence also occurs in the plant laccases and is
composed of the residues 535–555 in P. taeda laccase (Fig.
2B). Comparison of the X-ray crystal structure (Ducros et
al., 1998, 2001) and the sequence logo for the multi-copper
oxidases (Solomon et al., 1996) indicates that three of the
389
390
BIOTECHNOLOGY AND BIOENGINEERING, VOL. 83, NO. 4, AUGUST 20, 2003
Figure 2. Overview of the multiple sequence alignment for laccases. Eighty percent consensus sequence obtained after multiple sequence alignment of (A) 64 fungal laccase sequences; (B) 40
plant laccase sequences; (C) consensus sequence from (A) and (B). Multiple sequence alignment of protein sequences was carried out using ClustalW (Thompson et al., 1994). The conserved regions
are shaded if the length of the sub-sequence is greater than 7 residues. The corresponding regions, which are conserved both in plant and fungal laccases, are shaded in the same pattern. The starting
residue number for a given region is shown with reference to C. cinerius laccase in (A) and (C) and with reference P. taeda laccase in (B), while the length of the sub-sequences is shown in
parenthesis immediately below. Sequence logos (Schneider et al., 1990b) are shown for selected regions, L1–L4, which represent the frequency of occurrence of each amino acid at a particular
position in the ungapped region of the aligned protein sequences. Height of a letter in the logo represents the information content at that location.
Table II. Summary of conserved regions in the aligned amino acid sequences of fungal and plant laccases; sub-sequences are shown for all the ungapped
conserved regions that are at least three residues in length.

Start positiona Length Consensusb

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
Cu-ligating residues of the trinuclear copper cluster (T3
copper) (Fig. 2A) are housed within the L4 sequence logo.
In fungal laccases, Phe is the conserved residue at the axial
ligand position (residue 17 in the sequence logo), while in
plant laccases, Leu is the conserved residue at this position.
Minor differences between fungal and plant laccase L4
logos occur among the non-ligating residues.
In fungal laccases, the 21-residue L2 region near the N-
terminus (residues 104–124 of the C. cinerius laccase) also
conforms to the type I but not to the type II copper signature
sequence (Fig. 2A). This region houses two of the Cu-
ligating residues of the T3 copper center. Only the first eight
residues of this region, residues 136–143 of P. taeda lac-
case, are conserved in the plant laccases while the remaining
residues are highly variable (Fig. 2B). Thus, the 21-residue
L2 sequence is specific for the fungal laccases, while the L4
sequence is common to both the plant and fungal laccases.
Another large 24-residue conserved segment near the N-
terminus characterizes our L1 logo (Fig. 2). This logo, cor-
responding to residues 64–87 of C. cinerius laccase, houses
one of the T2 copper ligands and one of the T3 copper
ligands. An eight-residue conserved region, residues 396–
403 of C. cinerius laccase, houses one ligand each for the
T1, T2, and T3 copper ions (Fig. 2A). This region is con-
served in the plant laccases as well (Fig. 2B). In addition to
the conserved regions depicted in Figure 2, a few small
stretches, 3–7 residues in length, are conserved in the
aligned laccase sequences. A summary of these sequences is
shown in Table II. When mapped on to the X-ray crystal

Table III. Copper signature sequences.

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.

SURESH KUMAR ET AL.: ANALYSIS OF THE FUNGAL LACCASE FAMILY 391

structure of C. cinerius laccase (Fig. 1A), the conserved
sequences are found to be close in space, possibly forming
the enzyme catalytic apparatus and possibly the functional
interface for substrate binding. To best of our knowledge,
the functional significance of conserved residues in the
multi-copper oxidases has so far been reported for only the
residues that ligate the copper ions. Interestingly, the protein
can be divided into two distinct domains, with L1 and L2
housed in one domain and L3 and L4 housed in the other
domain (Fig. 1A). Some of the copper ions are located at the
interface of the two domains as reported previously.
Laccase Signature Sequences
We observe that the copper-ligating residues in the laccases
are housed in regions that are conserved across all laccases.
A total of 10 histidines and 1 cysteine are conserved across
all multi-copper oxidases and are thought to serve as the
copper ligands. In laccases, these residues are housed in the
four conserved regions that we have designated as signature
sequences L1–L4 (Table III). Of these, L2 and L4 are analo-
gous to the existing type I copper signature, while the L4
also conforms to the existing type II copper signature. The
remaining signatures L1 and L3 do not conform to any of
the known signature sequences for multi-copper oxidases.
The four signature regions L1–L4 together comprise the
specific signature for laccases, being more restrictive than
the existing signature sequences for the multi-copper oxi-
dases.
Comparison Between Laccases and Non-laccases
When the laccase sequence signature motifs, L1–L4, are
compared with the overall multi-copper oxidase sequence
signature, it becomes apparent that the residues that ligate
with the Cu ions are identical to the two signatures (data not
shown). This is in line with the findings of Solomon et al.
(1996) that were derived from alignments involving nine
multi-copper oxidase sequences. When equivalent regions
of other multi-copper oxidases were aligned to compare
with the L1–L4 signatures, the 80% consensus of multi-
copper oxidases showed only 1–3 residues conserved com-
pared to 6–14 conserved residues for the laccase signatures.
Thus, the L1–L4 signatures of laccases as shown in Table
III are indeed a unique feature of laccases distinguishing
them within the broad class of multi-copper oxidases.
Relationship Between L2 and L4 Sequences at
Structural and Sequence Levels
Close examination of the sequence logos L1–L4 reveals a
certain subtle similarity between the logos L1 and L3 and
between the logos L2 and L4. Specifically, the motif
HWHG in the logo L1 is equivalent to the motif HLHG in
the logo L3. Similarly, between the logos L2 and L4, Gly is
the common conserved residue at positions 1 and 16, while
His is the common conserved residue at positions 6 and 8.
392 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 83, NO. 4, AUGUST 20, 2003
Several other positions in the logos L2 and L4 feature amino
acids residues of similar polarity or similar conformational
propensity. Remarkably, there also are certain radical sub-
stitutions. For example, Gln at position 12 in logo L2 is
replaced by His at the equivalent position in logo L4. Con-
sidering these peculiar features of intra-protein similarity,
we suspect an event of structural duplication of functionally
important residues in laccase family. In fact, the sequence
similarity between the two corresponding regions, say be-
tween L2 and L4, of any given laccase sequence is statis-
tically not significant (data not shown). However, a column-
based alignment of two regions does highlight the individu-
ally conserved residues in the two regions.
Sequence Duplication, Evolution, and
Structural Conservation
Comparisons between L1 and L3 and between L2 and L4
establish that not only the copper-ligating residues are com-
pletely conserved in these logos, but there are several other
non-ligating residues that are either completely conserved
or semi-conserved, in being occupied by residues that have
a similar conformational propensity or a similar hydropathic
index. The pattern of conservation could thus have a con-
formational basis. In order to investigate such a possibility,
a comparison of L1 and L3 and that of L2 and L4 was made
in terms of the root mean square deviation (RMSD) between
their C␣ in the three-dimensional structure of the C. cinerius
laccase [PDB code 1A65] (Ducros et al., 1998). The RMSD
calculation (Holm and Sander, 1993) has established that
the conformational fold comprising the region 60–70 in the
protein is homologous to the conformational fold compris-
ing the region 395–495 in the protein (RMSD value of 1.18
Å). Similarly, the regions 102–127 and 444–469 in the pro-
tein share a significant conformational homology (RMSD
value of 1.15 Å) (Fig. 3A,B). The regions of conformational
homology are strictly localized since any residue extension
on either side of the designated regions in calculating the
RMSDs resulted in a sharp increase in the RMSD. A perusal
of the regions 60–127 (L1 and L2) and 395–469 (L3 and
L4) in the protein tertiary structure establishes a remarkable
conformational homology between them, in forming a quasi
C-2 symmetric core of the protein that characterizes the
active site region. In addition to the structural homology
between L1 and L3 and between L2 and L4, the relative
orientation of L1 with L2 is similar to that of L3 with L4
(Fig. 3C). It is possible that the symmetrical nature of this
active site architecture is the consequence of a gene dupli-
cation event, while the requirement of maintenance of this
unique three-dimensional fold in the vicinity of the Cu-
ligating residues possibly has acted as an evolutionary pres-
sure in maintaining residues with similar hydropathy or sec-
ondary structure propensity between the corresponding po-
sitions in L2 and L4. There are several telltale indications in
support such a conjuncture. The Gly residues at positions 1
and 16 in L2 and L4 occupy the right (lower)-half region of
the Ramchandran plot and, therefore, are stereochemically
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
event has been proposed earlier for the multi-copper oxidase
family based on domain recycling, but it has not been sup-
ported with sequence conservation pattern.
Based on analysis of three-dimensional structure of C.
cinerius laccase and the multiple sequence analysis of sev-
eral laccases, we propose that the residues that do not ligate
with copper ions were conserved/semi-conserved to main-
tain a local three-dimensional fold. In this report, we present
some of the hidden features of laccases, which are not ap-
parent by comparison of the amino acid sequences alone or
by comparison of the three-dimensional structures alone.
The possible gene duplication event is a cryptic one because
it is unrecognizable at the level of the sequence similarity
but is clearly implied by the observed conformation simi-
larity shared by the logos. Moreover, from an application
point of view, the sequence and structure motifs described
here could be used to search for new laccases and related
enzymes in newly sequenced genomes.
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