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ISSN 1063-7745, Crystallography Reports, 2006, Vol. 51, No. 5, pp. 817–823. © Pleiades Publishing, Inc., 2006.
Original Russian Text © A.V. Lyashenko, Yu.N. Zhukova, N.E. Zhukhlistova, V.N. Zaitsev, E.V. Stepanova, G.S. Kachalova, O.V. Koroleva, W. Voelter, Ch. Betzel, V.I. Tishkov,
I. Bento, A.G. Gabdulkhakov, E.Yu. Morgunova, P.F. Lindley, A.M. Mikhailov, 2006, published in Kristallografiya, 2006, Vol. 51, No. 5, pp. 870–877.
817
INTRODUCTION
Laccases belong to the blue oxidases (oxygen oxi-
doreductases, EC 1.10.3.2) with broad substrate speci-
ficity, which can be enhanced with the use of redox
mediators. Various phenols, such as mono-, di-, and
polyphenols, serve as substrates oxidized by laccase
[1–3]. These enzymes catalyze oxidation of substrate
molecules accompanied by reduction of molecular oxy-
gen to water. The
T
1 copper ion of the mononuclear
copper site serves as the primary electron acceptor. An
electron is transferred from the
T
1 copper ion to the tri-
nuclear site containing one
T
2 copper ion and two
T
3
copper ions (the
T
2/
T
3 site). The oxygen molecule
binds at the trinuclear site, where two water molecules
are generated after the four-electron transfer from four
substrate molecules. The classification of copper ions
(three types) in multinuclear copper-containing oxi-
dases (ascorbate oxidase, laccase, and ceruloplasmin)
is based on the differences in their spectral and mag-
netic properties [4–6]. The
T
1 copper ions (the mono-
nuclear site) are characterized by an absorption band
with a maximum at 610 nm and are responsible for the
blue color of concentrated solutions of these proteins.
The
T
2 copper ion (“non-blue copper”/EPR-active cop-
per) shows adsorption at 330 nm. The
T
3 copper ions
(EPR-inactive copper) exist as a binuclear cluster com-
posed of two antiferromagnetically coupled copper
atoms. The amino-acid residues binding the
T
1,
T
2, and
T
3 copper ions are strictly conserved in all representa-
tives of mononuclear copper-containing oxidases. The
first step of the proposed schemes of the evolution of
laccases involves the duplication of the common gene
precursor encoding the sequence of the blue pra protein
STRUCTURE
OF MACROMOLECULAR COMPOUNDS
Dedicated to the 60th Birthday of M.V. Kovalchuk
Three-Dimensional Structure
of Laccase from
Coriolus zonatus
at 2.6 Å Resolution
A. V. Lyashenko
a
, Yu. N. Zhukova
a
, N. E. Zhukhlistova
a
, V. N. Zaitsev
b
, E. V. Stepanova
c
,
G. S. Kachalova
d
, O. V. Koroleva
b
, W. Voelter
e
, Ch. Betzel
f
, V. I. Tishkov
g
, I. Bento
h
,
A. G. Gabdulkhakov
a, i
, E. Yu. Morgunova
a
, P. F. Lindley
h
, and A. M. Mikhailov
a
a
Shubnikov Institute of Crystallography, Russian Academy of Sciences, Leninski
œ
pr. 59, Moscow, 119333 Russia
e-mail: amm@ns.crys.ras.ru
b
Centre for Biomolecular Sciences, University of St. Andrews, North Haugh, St. Andrews, Fife KY16 9ST, UK
c
Bach Institute of Biochemistry, Russian Academy of Sciences, Leninski
œ
pr. 33, Moscow, 119991 Russia
d
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino,
Moscow oblast, 142290 Russia
e
Institute for Biochemistry, University of Tübingen, Germany
f
Department of Biochemistry and Molecular Biology, University of Hamburg, c/o DESY, Bld. 22a,
Notkestrasse 85, D-22603 Hamburg, Germany
g
Department of Chemical Enzymology, Moscow State University, Vorob’evy gory, Moscow, 119992 Russia
h
Institute for Technology in Chemistry and Biology, New University of Lisbon, 127
Av. da Republica, 2781-901 Oeiras, Portugal
i
Institute of Protein Research, Russian Academy of Sciences, Institutskaya ul. 4, Pushchino, Moscow oblast, 142290 Russia
Received May 5, 2006
Abstract
—Laccase (oxygen oxidoreductase, EC 1.14.18.1) belongs to the copper-containing oxidases. This
enzyme catalyzes reduction of molecular oxygen by different organic and inorganic compounds to water with-
out the formation of hydrogen peroxide. The three-dimensional structure of native laccase from
Coriolus zona-
tus
was solved and refined at 2.6 Å resolution (
R
factor
= 21.23%,
R
free
= 23.82%, rms deviations for the bond
lengths and bond angles are 0.008 Å and 1.19
°
, respectively). The primary structure of the polypeptide chain
and the architecture of the active site were refined. The carbohydrate component of the enzyme was identified.
The access and exit water channels providing the access of molecular oxygen to the active site and release of water,
which is the reduction product of molecular oxygen, from the protein molecule were found in the structure.
PACS numbers:
87.15.By
DOI:
10.1134/S1063774506050117
818
CRYSTALLOGRAPHY REPORTS
Vol. 51
No. 5
2006
LYASHENKO et al.
of the cupredoxin series containing mononuclear sites.
In the second step of evolution, the third domain is
incorporated and the trinuclear site is formed at the
interface between the first and third domains.
Laccase has a rather wide field of application in sci-
entific research (genetic engineering, studies of
biopolymer structures, etc.) to industrial processes
(biosensor technologies, organic synthesis, design of
new pharmaceuticals, bleaching of paper slurries and
cloths, detoxification of xenobiotics including pesti-
cides and nerve-paralytic agents, and stabilization of
beverages). In addition, owing to the ability of laccase
to catalyze electroreduction of molecular oxygen to
water molecules according to the mediatorless mecha-
nism, the kinetic and electrocatalytic properties of the
enzyme as a promising catalyst for electrode processes
have attracted considerable attention.
Laccases have been extensively studied by bio-
chemical and spectral methods and X-ray diffraction
[7–12], thereby providing insight into many structural
and functional features of these enzymes. Knowledge
of the three-dimensional structures of laccases from
different sources and the elucidation of the mechanism
of catalysis are necessary for the more successful prac-
tical application of the enzyme. The present study is a
continuation of X-ray diffraction investigations of
fungi laccase from
Coriolus zonatus
. The results of the
preliminary investigation have been reported in [13].
The aim of the present study was to refine the three-
dimensional organization of laccase from
Coriolus
zonatus
, its active site, water channels, the carbohy-
drate component, and the primary sequence of this
enzyme by X-ray diffraction methods.
EXPERIMENTAL
Isolation and purification of laccase from
C. zonatus.
Coriolus zonatus
from the collection of the
Bach Institute of Biochemistry of the Russian Academy
of Sciences (
GenBank Accession number Trametes
ochracea
—AB158314) was used as the producer strain
of laccase. The strain was preserved on agarized media,
which were prepared through dilution of a wart with
water in a 1 : 4 ratio (v/v) in the presence of 2% agar at
+4
°
C with a colorless reverse. The sowing material was
grown by the surface method in a nutrient medium at
initial pH 6.0 containing peptone as the nitrogen
source, glucose as the carbon source, and mineral salts
at 25–27
°
C. The following medium was used in all exper-
iments (g/l): glucose, 10.0; peptone, 3.0; KH
2
PO
4
, 0.6;
ZnSO
4
· 7H
2
O, 0.001; K
2
HPO
4
, 0.4; FeSO
4
· 7H
2
O,
0.0005; MnSO
4
, 0.05; and MgSO
4
· 7H
2
O, 0.5. The
deep cultivation of the producer of the fungus
Coriolus
zonatus
was carried out as follows: CuSO
4
(0.15 g/l)
and CaCl
2
(0.5 g/l) were added to the nutrient medium
of the above-mentioned composition, and pH was
adjusted to 6.0 with a NaOH solution or acetic acid.
The medium was sterilized in an autoclave at 1 atm for
30 min. After completion of cultivation, the culture fil-
trate was separated from micelles by filtration and deep
freezing was performed at –40
°
C.
Extracellular laccase from
Coriolus zonatus
was
isolated from the culture liquid (pH 5.0) at room tem-
perature by precipitation with 90% ammonium sulfate
for 2 h with constant stirring. The precipitate was col-
lected by centrifugation at 2500
g
for 30 min and again
dissolved in a minimum volume of distilled water. Then
the sample was applied to a Sephadex G-25 column
(100
×
2 cm) equilibrated with a 5 mM potassium phos-
phate buffer (PPB), pH 6.0. The elution was carried out
with the use of the same buffer. The peak containing the
enzymatic activity was applied to a DEAE-Toyopearl
650 M column (2
×
20 cm) equilibrated with 5 mM
PPB, pH 6.0. The protein was eluted with a linear ionic-
strength gradient of PPB (2
×
150 ml), pH 7.2, and the
molarity was varied from 5 to 200 mM. The active frac-
tions were combined, dialyzed against 5 mM PPB, pH
6.0, and rechromatographed on a DEAE-Toyopearl
650 M column under the same conditions but with a
more gently sloping ionic-strength gradient (2
×
250 ml). A highly homogeneous sample of the enzyme
required for crystallization was prepared by high per-
formance liquid chromatography (HPLC) on an FPLC
chromatograph using a TSK 3000 gel-filtration col-
umn. The elution was performed with 50 mM PPB, pH
6.5, at a rate of 0.5 ml/h. The homogeneity of the sam-
ple was checked by electrophoresis (Fig. 1).
Crystallization.
Crystals of laccase from
C. zona-
tus
(Fig. 2) were grown by the hanging-drop vapor-dif-
fusion method. The reservoir solution (0.5 ml) con-
tained 0.2 M ammonium sulfate, 0.1 M sodium acetate
(pH 4.6), and 25% (
w/
v
) polyethylene glycol 4000. The
drops (6
µ
l) were composed of the protein (8 mg/ml) in
a 50 mM citrate buffer (pH 5.5), 0.1 M ammonium sul-
fate, 0.05 M sodium acetate (pH 4.6), and 12.5% (
w
/
v
)
polyethylene glycol 4000.
X-ray diffraction data collection.
X-ray intensities
were measured from crystals of laccase from
C. zona-
tus
at 100 K on a BW6 beamline (DESY, Hamburg,
Germany, a CCD detector) at a wavelength of 1.05 Å.
The reservoir solution, to which 15% glycerol (v/v) was
added, was used as a cryosolution. The experimental
structure amplitudes were obtained with the use of the
XDS program package [14]. The crystallographic char-
acteristics of the X-ray data set for laccase from
C. zonatus
and details of X-ray data collection are sum-
marized in Table 1.
Determination and refinement of the three-
dimensional structure of laccase.
The crystal struc-
ture was solved by the molecular replacement method
using the MOLREP program [15] incorporated in the
CCP4 suite of programs [16]. The three-dimensional
structure of laccase from
C. zonatus
, which we have
established earlier at 3.2 Å resolution [13], was used as
the starting model for the structure solution and refine-
ment. The water molecules, copper atoms of the active
CRYSTALLOGRAPHY REPORTS
Vol. 51
No. 5
2006
THREE-DIMENSIONAL STRUCTURE 819
site, and carbohydrates of laccase were excluded from
the starting model. The true solution corresponded to
rotation- and translation-function maxima, for which
the
R
fact
and
R
corr
were 38.7 and 63.2, respectively
(Fig. 3).
The structure was refined with the use of the CNS
[17] and REFMAC [18] programs. The refinement was
monitored with the use of the free
R
factor (
R
free
) calcu-
lated for 5% of reflections, which were arbitrarily cho-
sen from the experimental X-ray data. The manual
rebuilding was performed with the use of the O [19] and
COOT [20] programs based on
(3
F
o
– 2
F
c
), (2
F
o
–
F
c
)
,
and
(Fo – Fc) difference Fourier maps, where Fo and Fc
are the observed and calculated structure amplitudes,
respectively. The average B factors for the structure
were calculated with the Baverage program incorpo-
rated in the CCP4 suite of programs.
The correctness of the results in the course of refine-
ment was monitored with the Procheck [21],
WhatCheck [22], and COOT [20] programs. The
refinement statistics for the structure of laccase from C.
zonatus are given in Table 2.
RESULTS AND DISCUSSION
Laccase from C. zonatus is a single-chain molecule
consisting of 499 amino-acid residues. The primary
structure of the enzyme, which was determined on the
basis of results of the present X-ray diffraction study, is
shown in Fig. 4. A comparison with the primary struc-
ture of laccase from T. versicolor (ID PDB 1GYC),
which we have used as the starting model in the early
steps of X-ray diffraction study of laccase from
C. zonatus [13], revealed 123 different amino-acid res-
idues.
The laccase under consideration exists as a mono-
mer (Fig. 5) consisting of three cupredoxin domains.
The first domain includes residues from 1 to 131 and
contains one helix and seven β-strands, the second
domain includes residues from 132 to 309 and consists
of eleven β-strands, and the third domain includes resi-
dues from 310 to 499 and contains three helices and six
β-strands. The first domain is linked to the second and
third domains by two disulfide bridges (Cys117–
Cys205, 2.03 Å; and Cys85–Cys488, 2.04 Å).
Structure of the active site. The copper atoms of
the active site were observed as the following four high-
est-density peaks in (Fo – Fc) difference electron den-
sity maps: Cu(1), 7.0σ; Cu(2) and Cu(3), 10.5σ; and
Cu(4), 7.3σ, where σ is the rms error of the electron
density map. The T2 and T3 copper atoms form a trinu-
clear cluster located between domains I and III (Fig. 6).
93.0
67.0
43.0
30.0
20.1
14.1
Molecular weight, kDa
Fig. 1. SDS-electophoretic pattern for laccase from C. zona-
tus (to the right of the figure) and marker proteins with the
molecular weights in kDa (to the left).
0.5 mm
Fig. 2. Crystals of laccases from C. zonatus.
Table 1. Crystallographic data and details of X-ray data col-
lection for laccase from C. zonatus
Sp. gr. P321 (No. 150)
Unit-cell parameters, Å, deg a = b = 168.93, c = 69.35
α = β = 90.0, γ = 120.0
Molecular weight, kDa 60
Number of molecules per asym-
metric unit 1
Wavelength, Å 1.05
Resolution, Å 145.86–2.60
Number of measured reflections 175868
Crystal-to-detector distance, mm 180
Oscillation range, deg 0.5
Rotation range, deg 80
Number of independent reflections 35011
Redundancy 5.02 (4.75)*
Completeness of the set, % 95.07
Mosaicity, deg 0.2
Average I/σ(I) 15.17 (4.27)*
Rmerge 7.1 (24.4)*
Matthews coefficient (VM), Å3 Da–1 4.8
Solvent content, % 74
* High-resolution data are given in parentheses.
820
CRYSTALLOGRAPHY REPORTS Vol. 51 No. 5 2006
LYASHENKO et al.
The T1 copper atom is located in domain III in the
vicinity of the substrate-binding site analogously to that
found in other multinuclear copper-containing oxi-
dases. The Cu(1) atom is coordinated by two histidine
residues (His395 and His458) and a cysteine residue
(Cys453) lying in a plane with the copper ion (Table 3,
Fig. 6). The axial position on the side of the substrate
pocket is occupied by Ile455, which forms the shortest
contact (3.74 Å) with the T1 copper atom. The amino-
acid residue Phe463 at a distance of 3.62 Å from Cu(1)
is located on the opposite side. Other fungi laccases
also contain aliphatic residues in the axial positions.
For example, the axial position in laccase from C.
cinereus is occupied by the residue Leu462 at a dis-
tance of 3.51 Å from the copper ion. In laccase from T.
versicolor, the corresponding position is occupied by
phenylalanine at a distance of 3.6 Å [7, 8]. This three-
dimensional configuration of the T1 site differs from
that found in ascorbate oxidase, laccase cotA from
Bacillus subtilis, and laccase CueO from Escherichia
coli, in which the sulfur atom of methionine occupies
the axial position at a distance of approximately 3.0 Å
from copper [23–25].
Eight histidines (four amino-acid residues from
domain I and four residues from domain III) are
involved in coordination of the copper atoms of the tri-
nuclear (the T2/T3 cluster) site (Fig. 6). The Cu(2) and
Cu(3) atoms are coordinated by six histidines (each
copper atom is coordinated by three amino-acid resi-
dues). The NE2 atoms of five histidines are involved in
coordination, whereas the sixth histidine is involved in
coordination through the ND1 atom (Table 3). The dis-
tance between the copper atoms, Cu(2)–Cu(3), is
4.80 Å. An electron-density peak asymmetrically
related to the Cu(2) and Cu(3) atoms is observed in
(Fo – Fc) difference maps between these copper atoms.
The careful refinement made it possible to identify this
peak as an oxygen atom of hydroxyl or water (Table 3).
The Cu(2)–O–Cu(3) angle is 156.25°. As a result, the
70%
60%
50%
40%
30%
20%
10%
0% 12345678910
Fig. 3. Graphical representation of the molecular-replace-
ment solution in searching for the starting model for laccase
from C. zonatus with the use of the MOLREP program [14],
where the gray and black columns are Rcorr and Rfact,
respectively.
1–60
61–120
121–180
181–240
241–300
301–360
361–420
421–480
481–499
S1S2S3S4
S5S6h1
S7S8S9
S10 S11 S12 S13 S14 S15
S16 S17 S18
S19 h2
S20 S21
S22 S23 h3S24
h4
Fig. 4. Primary sequence of laccase from C. zonatus and secondary-structure elements, where S is a β-ribbon and h is a helix.
CRYSTALLOGRAPHY REPORTS Vol. 51 No. 5 2006
THREE-DIMENSIONAL STRUCTURE 821
coordination sphere of each copper atom can be
described as a distorted tetrahedron. The Cu(2) and
Cu(3) atoms are located from the Cu(4) atom of the T2
type at distances of 4.29 and 3.91 Å, respectively. The
Cu(4) atom is coordinated by two histidines (Table 3).
A peak found at a distance of 2.79 Å from the latter
copper atom was refined as a water molecule. This
water molecule and two histidine residues form a trigo-
nal planar configuration of the T2 site.
The T1 copper atom is linked to the trinuclear site by
the tripeptide His452–Cys453–His454, which is con-
served in all multinuclear copper oxidases (Table 3,
Fig. 6). The distances from the Cu(1) atom (T1 type) to
the Cu(2), Cu(3) (T3 type), and Cu(4) (T2 type) atoms
are 12.02, 13.12, and 14.68 Å, respectively. The possi-
ble electron-transfer path between the mononuclear site
and the trinuclear sites has been proposed for the first
time for ascorbate oxidase (AO) [23]. The structural
similarity of the active sites of AO and laccase implies
the similarity of the electron-transfer mechanisms in
these proteins. By analogy with AO, the sulfur atom of
Cys453 in the coordination sphere of the T1 copper
atom in laccase from C. zonatus accepts an electron
from the Cu(+1) ion, which is, correspondingly, oxi-
dized to Cu(+2). Then, the electron is successively
transferred to the carbonyl oxygen atom of Cys453 and,
through a hydrogen bond, to the ND1 atom of His452,
the latter serving as a ligand of Cu(3) in the trinuclear
site (Fig. 6). The length of the
O_Cys453····ND1_His452 hydrogen bond involved in
the electron transport from the mononuclear site (the T1
copper ion) to the trinuclear (the T2/T3 cluster) site is
2.76 Å.
Water channels. In the molecule of laccase from
C. zonatus, two water channels leading to the trinuclear
site were revealed (Figs. 5 and 7). The water molecules
in these channels are involved in numerous hydrogen
bonds with each other and with the amino-acid residues
lining the walls of the channels. In one channel (Fig. 7),
which provides the access of oxygen molecules to the
T3 copper ions, the O_2, O_61, O_62, O_103, O_41,
and O_58 water molecules were located in difference
electron density maps. These water molecules are
linked to each other by hydrogen bonds (the distances
vary from 2.53 to 3.19 Å). The O_2 water molecule is
linked to the O_61 water molecule in the channel, is
located asymmetrically between the T3 copper ions,
and serves as a bridge (Fig. 7). Most amino-acid resi-
dues lining the walls of this channel, such as Ala80,
Ser110, His111, Ser113, and Ty r116 from domain I and
His454 and Asp456 from domain III are conserved in
the laccase family. These residues are involved in the
following hydrogen bonds with the water molecules
occupying the first channel: O_His111···O_41, 3.38 Å;
NE2_His111···O_61, 3.01 Å; O_Ser113···O_103, 2.77 Å;
N–Ser113···O_103, 3.13 Å; N_Ser113···O_58, 3.13 Å;
NE2_His454···O_61, 3.00 Å; and OD1_Asp456···O62,
2.55 Å.
Another water channel (Figs. 5 and 7) serves for the
transport of water molecules from the T2 site to the sur-
face of the protein molecule. The O_7, O_113, O_69,
O_21, and O_28 molecules form a chain and are
located at distances of 2.55–3.39 Å from each other.
Like the amino-acid residues of the first channel, the
residues of the second channel, such as Gly67 and
Gln102 of domain I, Asp 224 of domain II, and Leu399,
His402, and Asp424 of domain III, are conserved in the
laccase family and are linked to the water molecules in
the channels by a hydrogen bond network. The main-
chain oxygen atoms of Leu399 and His402 are involved
in hydrogen bonds with the O_113 water molecule
(O_His402···O_113, 3.08 Å; O_Leu399···O_113,
3.01 Å), which is, in turn, located at a hydrogen-bond
distance (2.78 Å) from the water molecule involved in
the coordination sphere of the T2 copper ion (2.79 Å).
The O_69 water molecule forms short contacts with
O_Gly67 (3.01 Å) and OD1_Asp424 (2.6 Å). In addi-
tion, two water molecules, O_21 and O_28, from the
second channel, which are linked to each other by a
hydrogen bond (2.74 Å), are involved in hydrogen
bonds with O_Asp224 (2.95 Å) and NE2_Gln102
(2.96 Å), respectively. Analogous access and exit water
channels were found in the structures of other laccases
as well [9, 24, 26].
Table 2. Refinement statistics for the structure of laccase
Crystal laccase from
C. zonatus
Resolution range, Å 29.0–2.6 (2.62–2.60)*
Number of reflections in the working set 31 805 (2289)*
Number of reflections in the test set (5%) 1691 (116)*
Cutoff of the set, F > σ
Number of non-hydrogen atoms of the
protein molecule 3899
Number of water molecules 117
Number of carbohydrate molecules:
NAG 4
MAN 1
Rfact, % 21.23 (31.8)*
Rfree, % 23.82 (36.8)*
rms deviation for bond lengths, Å 0.008
rms deviation for bond angles, deg 1.19
Average B factor, Å2: 30.405
for main-chain atoms 30.369
for side-chain atoms and water mole-
cules 39.102
Ramachandran statistics, %:
most favored region 84.7
allowed region 14.8
additionally allowed region 0.2
disallowed region 0.2
822
CRYSTALLOGRAPHY REPORTS Vol. 51 No. 5 2006
LYASHENKO et al.
Carbohydrate component of laccase. Laccase,
like all blue oxidases, is a glycoprotein. The carbohy-
drate component of most laccases comprises 10–13%
of the weight of the protein molecule and includes such
carbohydrates as hexosamine, glucose, mannose,
galactose, and arabinose. High-electron-density
regions, which were found in difference-electron-den-
sity maps at the periphery of the molecule in the vicin-
ity of the amino-acid residues Asn54, Asn333, and
Asn436, were interpreted as carbohydrates (Fig. 5).
One of the electron-density regions was interpreted as
two N-acetylgalactosamines (NAG5 and NAG1), which
are covalently bonded to each other (the bond length is
1.35 Å) and to ND2_Asn54 (NAG5; the bond length is
1.35 Å). In addition, the NAG5 molecule is involved in
the SD_Met57···O7_NAG5 hydrogen bond (2.65 Å)
with the amino-acid residue Met57. The O4 atom of N-
acetylgalactosamine NAG2 is located at a hydrogen-
bond distance (2.31 Å) from ND2_Asn333. The amino-
acid residue Asn436 is involved in a covalent bond with
the mannose molecule MAN3, which is, in turn,
covalently bonded to the NAG4 molecule.
Heterogeneity of the carbohydrate component and
its disorder in protein molecules always present great
problems in growing crystals of laccases suitable for
X-ray diffraction. Owing to the lability of carbohydrate
chains, glycan residues generally cannot be located in
electron density maps. Evidently, the covalent bonds
between of the carbohydrate chains found in the struc-
ture of laccase from C. zonatus facilitate ordering of the
carbohydrate component in the structure, and the for-
mation of these bonds plays, apparently, an important
role in the growth of stable and ordered crystals of this
protein.
Mechanism of action of the enzyme. The mecha-
nism of action of multimedia oxidases has been pro-
posed in [26]. The hypothesis of the authors was based
on X-ray diffraction studies of the crystal structures of
laccase complexes, in which molecular oxygen, perox-
ide, or azide occupy the bridging positions between the
T3 copper ions. The structure of laccase from C. zona-
tus is yet another “instant photograph,” which fixes one
of the possible events of the enzymatic reaction. After
the transformation of molecular oxygen into two
hydroxide ions, one of the ions is protonated and then
migrates as a water molecule into the exit channel, the
second hydroxide ion remaining bound to one of the T3
copper atoms. After protonation of this hydroxide ion,
the second water molecule migrates to the exit channel.
The formation of two water molecules occurs after the
four-electron transfer from four substrate molecules.
Apparently, the amino-acid residues lining the walls of
the access channel serve as the proton source. This
mechanism assumes a considerable mobility of the
Domain III
Access
Domain I
Exit
Domain II
S19 S20
S21 S24 h2
h4
h1
N
S22 S23
S5
S6
S4
S6
S7S1
S3S9
S2
S11
S16 S17 S18
S10
S8
S10
Fig. 5. Tertiary structure of laccase from C. zonatus.
His64
His66
His400
His398 His395
Cu(1)
Cu(2)
Cu(3)
Cu(4)
Cys453
His458
His454
His111
His109
His452
H2O
H2O
Fig. 6. Structure of the active site of laccase from
C. zonatus.
His402
His454
His111
Ser113
Asp456 Asp424
Asp224
Gly67
Gln102
Cu(2)
Cu(4)
Cu(3)
Leu399
Fig. 7. Three-dimensional structure of the water channels
including water molecules (filled circles).
CRYSTALLOGRAPHY REPORTS Vol. 51 No. 5 2006
THREE-DIMENSIONAL STRUCTURE 823
Cu(4) ion of the T2 type because it should provide the
access of the hydroxide ions to the exit channel. The
less rigid configuration of the T2 copper atoms relative
to the T3 copper atom allows higher mobility of the
Cu(4) ion. This finding is consistent with the experi-
mental observations. Thus, purified samples of laccases
from different sources often lose T2 copper atoms
either completely or partially.
ACKNOWLEDGMENTS
We thank the Komarov Botanical Institute of the
Russian Academy of Sciences for providing a producer
strain of extracellular laccase from Coriolus zonatus.
This study was supported by the BMFW
(Bundesministerium für Forschung und Wissenschaft,
contract No. RUS/214) and in part by the Russian
Foundation for Basic Research (project no. 05-04-
49520).
REFERENCES
1. A. I. Yaropolov, O. V. Skorobogat’ko, S. S. Vartanov, and
S. D. Varfolomeyev, Appl. Biochem. Biotechnol. 49 (3),
257 (1994).
2. D. R. McMillin and M. K. Eggleston, in Multi-Copper
Oxidases, Ed. by A. Messerschmidt (World Scientific
Publishing Co. Pte. Ltd, 1997), Chap. 5, p. 129.
3. B. Reinhammar, in Copper Proteins and Copper
Enzymes, Ed. by R. Lontie (CRC Press, Boca Raton,
1984), Vol. 3, p. 1.
4. B. G. Malmstrom, Annu. Rev. Biochem. 51, 21 (1982).
5. B. G. Malmstrom, in Multi-Copper Oxidases, Ed. by
A. Messerschmidt (World Scientific Publishing Co. Pte.
Ltd, 1997), p. 1.
6. K. Nakamura and N. Go, Cellular and Molecular Life
Sciences (Birkhauser, Bassel, 2005), p. 1.
7. V. Ducros, A. M. Brzozowski, K. S. Wilson, et al., Nat.
Struct. Biol. 5, 310 (1998).
8. V. Ducros, A. M. Brzozowski, K. S. Wilson, et al., Acta
Crystallogr., Sect. D: Biol. Crystallogr. 57, 333 (2001).
9. K. Piontek, M. Antorini, and T. Choinowski, J. Biol.
Chem. 277 (40), 37663 (2002).
10. T. Bertrand, C. Jolivalt, P. Briozzo, et al., Biochemistry
41, 7325 (2002).
11. N. Hakulinen, L. L. Kiiskinen, K. Kruus, et al., Nat.
Struct. Biol. 9, 601 (2002).
12. F. J. Enguita, D. Marcal, L. O. Martins, et al., J. Biol.
Chem. 279, 23 472 (2004).
13. A. V. Lyashenko, N. E. Zhukhlistova, E. V. Stepanova,
et al., Kristallografiya 51 (2), 305 (2006) [Crystallogr.
Rep. 51, 278 (2006)].
14. W. Kabsch, International Tables for Crystallography,
Ed. by M. G. Rossmann and E. Arnold (Kluwer, Dor-
drecht, 2001).
15. A. A. Vaguine, J. Richelle, and S. J. Wodak, Acta Crys-
tallogr., Sect. D: Biol. Crystallogr. 55, 191 (1999).
16. Collaborative Computational Project, Number 4, Acta
Crystallogr., Sect. D: Biol. Crystallogr. 50, 760 (1994).
17. A. T. Brunger, P. D. Adams, and G. M. Clore, Acta Crys-
tallogr., Sect. D: Biol. Crystallogr. 54, 905 (1998).
18. G. N. Murshudov, A. A. Vagin, and A. Lebedev, Acta
Crystallogr., Sect. D: Biol. Crystallogr. 55, 247 (1999).
19. T. A. Jones, J.-Y. Zou, S. W. Cowan, and M. Kjeldgaard,
Acta Cristallogr., Sect. A: Found Crystallogr. 47, 110
(1991).
20. P. Emsley and K. Cowtan, Acta Crystallogr., Sect. D:
Biol. Crystallogr. 60, 2126 (2004).
21. R. A. Laskowski, M. W. MacArthur, D. S. Moss, and
J. M. Thornton J. Appl. Crystallogr. 26, 283 (1993).
22. R. Rodriguez, G. Chinea, N. Lopez, et al., CABIOS,
Comput. Appl. Biosci., No. 14, 523 (1984).
23. A. Messerchmidt, R. Ladenstein, and R. Huber, J. Mol.
Biol. 224, 179 (1992).
24. F. J. Enguita, L. O. Martins, A. O. Henriques, and
M. A. Carrondo, J. Biol. Chem. 278, 19 416 (2003).
25. S. A. Roberts, A. Weichsel, and G. Grass, Proc. Natl.
Acad. Sci. U.S.A. 99, 2766 (2002).
26. I. Bento, L. O. Martins, and G. G. Lopes, Dalton Trans.,
3507 (2005).
Translated by T. Safonova
Table 3. Distances between the copper atoms and ligands
Type of the
copper atom Atom Atom_amino-acid
residue Distance,
Å
T1 Cu(1) ND1_His395 2.47
Cu(1) S_Cys453 2.11
Cu(1) ND1_His458 2.39
T3 Cu(2) NE2_His111 2.03
Cu(2) NE2_His400 2.03
Cu(2) NE2_His452 1.96
Cu(2) O_2 1.93
T3 Cu(3) ND1_His66 2.01
Cu(3) NE2_His109 2.19
Cu(3) NE2_His454 2.18
Cu(3) O_2 2.96
T2 Cu(4) NE2_His64 1.93
Cu(4) NE2_His398 1.91
