CcCel6C consists of a dis-torted seven-stranded b⁄ a barrel and has an enclosed tunnel, which is observed in other cellobiohydrolases from ascomecetes Hypocrea jecorina HjeCel6A and Humi
Trang 1enzyme, CcCel6C, a cellulase constitutively produced by Coprinopsis cinerea
Yuan Liu1, Makoto Yoshida1, Yuma Kurakata2, Takatsugu Miyazaki2, Kiyohiko Igarashi3,
Masahiro Samejima3, Kiyoharu Fukuda1, Atsushi Nishikawa2 and Takashi Tonozuka2
1 Department of Environmental and Natural Resource Science, Tokyo University of Agriculture and Technology, Japan
2 Department of Applied Biological Science, Tokyo University of Agriculture and Technology, Japan
3 Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan
Introduction
Cellulose, a linear polymer made up of glucose units
linked by b-1,4-glucosidic linkages, is the predominant
structural component of plant cell walls and is the
most abundant biomass resource on Earth Cellulases
hydrolyse the b-1,4-glucosidic bonds of cellulose chains
and are traditionally classified as endoglucanases (EC
3.2.1.4) or cellobiohydrolases (EC 3.2.1.91) based on
their activity profiles Endoglucanases randomly cleave
the internal b-1,4-glucosidic bond of cellulose, whereas
cellobiohydrolases preferentially act on the end of the
chain and progressively cleave off cellobiose as the
main product [1–3]
Cellulases belonging to the glycoside hydrolase family
6 (GH6) are known as major cellulolytic enzymes pro-duced by filamentous fungi For example, GH6 cellulases from the best studied cellulolytic organism, ascomycete Hypocrea jecorina(formerly known as Trichoderma ree-sei), make up 12–20% of total extracellular protein when the fungus grows in cellulolytic culture [4] Therefore, GH6 enzymes have been considered attractive enzymes for industrial application, such as biomass conversion The CAZy database (http://www.cazy.org/) [5] broadly categorizes the GH6 enzymes into cellobiohydrolase-type and endoglucanase-cellobiohydrolase-type enzymes In 1990, the first
Keywords
basidiomycete; cellobiohydrolase; cellulase
induction; endoglucanase; glycoside
hydrolase family 6
Correspondence
T Tonozuka, Department of Applied
Biological Science, Tokyo University of
Agriculture and Technology, 3-5-8
Saiwai-cho, Fuchu, Tokyo 183-8509, Japan
Fax: +81 42 367 5705
Tel: +81 42 367 5702
E-mail: tonozuka@cc.tuat.ac.jp
(Received 18 November 2009, revised 4
January 2010, accepted 14 January 2010)
doi:10.1111/j.1742-4658.2010.07582.x
The basidiomycete Coprinopsis cinerea produces the glycoside hydrolase family 6 enzyme CcCel6C at low and constitutive levels CcCel6C exhibits unusual cellobiohydrolase activity; it hydrolyses carboxymethyl cellulose, which is a poor substrate for typical cellobiohydrolases Here, we deter-mined the crystal structures of CcCel6C unbound and in complex with p-nitrophenyl b-d-cellotrioside and cellobiose CcCel6C consists of a dis-torted seven-stranded b⁄ a barrel and has an enclosed tunnel, which is observed in other cellobiohydrolases from ascomecetes Hypocrea jecorina (HjeCel6A) and Humicola insolens (HinCel6A) In HjeCel6A and HinCel6A, ligand binding produces a conformational change that narrows this tunnel In contrast, the tunnel remains wide in CcCel6C and the con-formational change appears to be less favourable than in HjeCel6A and HinCel6A The ligand binding cleft for subsite)3 of CcCel6C is also wide and is rather similar to that of endoglucanase These results suggest that the open tunnel and the wide cleft are suitable for the hydrolysis of carb-oxymethyl cellulose
Abbreviations
CcCel6C, Coprinopsis cinerea Cel6C; GH, glycoside hydrolase family; (Glc)2-S-(Glc)2, methylcellobiosyl-4-thio-b-cellobioside; HinCel6A, Humicola insolens Cel6A; HinCel6B, Humicola insolens Cel6B; HjeCel6A, Hypocrea jecorina Cel6A; PcCel7A, Phanerochaete chrysosporium, Cel7A; pNPG2, p-nitrophenyl b- D -cellobioside; pNPG3, p-nitrophenyl b- D -cellotrioside.
Trang 2crystal structure of a cellulase was reported; it was
a catalytic domain of Hypocrea jecorina Cel6A
(HjeCel6A, formerly designated cellobiohydrolase II),
a GH6 cellobiohydrolase from an ascomycete [6] In
that same decade, the crystal structure of the catalytic
domain in HinCel6A, another GH6 cellobiohydrolase
from ascomycete Humicola insolens, was determined
[7] The catalytic domains of HjeCel6A and HinCel6A
consist of a distorted seven-stranded b⁄ a barrel; the
striking feature is that they have active sites enclosed
by N-terminal and C-terminal loops that form a
tunnel The enclosed active sites trap the cellulose
chain in the tunnel and delay enzyme–substrate
dissociation, which promotes the cleavage of several
sequential substrate bonds [8,9] In contrast and
despite displaying higher sequence similarity to
HjeCel6A and HinCel6A, the structure of a fungal
endoglucanase, Humicola insolens Cel6B (HinCel6B),
shows active sites in a cleft formed by a C-terminal
loop deletion coupled with the peeling open of an
N-terminal loop [10]
Many reports are available on the crystal structures
of the GH6 enzymes from ascomycetes, but no crystal
structure of the basidiomycete-derived GH6 enzyme
has yet been determined Recently, we cloned five
genes encoding GH6 enzymes from a basidiomycete
Coprinopsis cinerea (formerly known as Coprinus
cine-reus), and the enzymes have been designated CcCel6A,
CcCel6B, CcCel6C, CcCel6D and CcCel6E [11] The
amino acid sequences corresponding to the active site
enclosing loops of cellobiohydrolases have been
observed in all five enzymes In the evolutionary tree,
however, four of the enzymes, CcCel6B–6E, have
mapped to a region distant from CcCel6A There are
high sequence identities of CcCel6A–HjeCel6A (48%)
and CcCel6A–HinCel6A (52%), including an
N-termi-nal cellulose binding domain In contrast, CcCel6B–6E
fall into a region closer to the endoglucanase
HinCel6B in the evolutionary tree, and no cellulose
binding domain is found in the four enzymes For
example, the sequence identities of CcCel6C–HjeCel6A
and CcCel6C–HinCel6A are 36 and 39%, respectively,
whereas that of CcCel6C–HinCel6B is 43% Transcript
analysis showed that the presence of cellobiose
strongly induced transcription of the CcCel6A gene,
but weakly induced transcription of the CcCel6B, -6D
and -6E genes Interestingly, the transcript level of
CcCel6C was not influenced by either glucose or
cellobiose When the enzymatic properties were
investigated, CcCel6B and CcCel6C exhibited
cellobio-hydrolase activity, but the enzymes hydrolysed
carb-oxymethyl cellulose, which is a poor substrate for
typical GH6 cellobiohydrolases [12] These results
indi-cate that the physiological function and the substrate binding mechanism of CcCel6C are expected to be dif-ferent from those of known cellobiohydrolases Here,
we present the crystal structure of CcCel6C To our knowledge, this is the first report of the crystal struc-ture of a basidiomycete GH6 enzyme
Results and Discussion
Overall structures of CcCel6C The crystal structures of unliganded CcCel6C and the enzyme–substrate complexes of CcCel6C–p-nitrophenyl b-d-cellotrioside (pNPG3) and CcCel6C–cellobiose were determined at 1.6, 1.4 and 1.2 A˚ resolutions, respectively (Table 1) The crystal belongs to the space group P1, which contains one molecule in an asymmetric unit In Ramachandran plots, 95.7% (unli-ganded CcCel6C), 95.7% (CcCel6C–pNPG3) and 95.4% (CcCel6C–cellobiose) of residues were shown to
be in favoured regions, and no residues were identified
as outliers, as calculated by the molprobity server [13] The electron density (2Fo–Fc) maps contoured at 1r show continuous density for almost all main chain atoms except for the first 12 N-terminal residues and the last 12 C-terminal segments containing the His-tag sequence The overall structure of CcCel6C alone is shown in Fig 1A Like the fungal GH6 cello-biohydrolases [6,7], CcCel6C consists of a seven-stranded b⁄ a barrel fold a-Helices and b-strands are numbered as a1–a8and b0–bVII, respectively, as shown
in Fig 2, based on the numbering scheme for HinCel6A [7]
Structural homology was researched using the dali server [14] and CcCel6C was found to most resemble the fungal GH6 enzymes: HinCel6A (cellobiohydro-lase; Z score, 54.2) [7], HjeCel6A (cellobiohydro(cellobiohydro-lase;
Z score 54.1) [6], HinCel6B (endoglucanase; Z score, 51.1) [10] and bacterial GH6 enzymes (e.g 1UOZ [15] and 1TML [16]; Z score 30) The Ca backbone of unliganded CcCel6C was superposed with those of cellobiohydrolases HjeCel6A, HinCel6A and endoglu-canase HinCel6B using the program superpose in the ccp4 suite [17] The results indicated that the folds of CcCel6C are almost identical to not only the cellobio-hydrolases, but also to the endoglucanase (Fig 1B) The rmsd values are 1.16 A˚ (CcCel6C–HinCel6A, 1BVW), 1.14 A˚ (CcCel6C–HjeCel6A, 1QK0 chain A) and 1.30 A˚ (CcCel6C–HinCel6B, 1DYS chain A) for main chain atoms
The significant feature in cellobiohydrolases HjeCel6A and HinCel6A is their active site located inside an enclosed tunnel [6–10,18]; CcCel6C has a
Trang 3homologous tunnel and its conformation is very
similar in the liganded and unliganded enzymes
(Fig 1C) Two loops, loop-1 and loop-2, forming this
tunnel are identified between bII and a4 and between
bVII and a8’ (Fig 2) Loop-1 and loop-2 contain
disulfide bridges of Cys103–Cys164 and Cys298–
Cys348, respectively, like those seen in HjeCel6A and
HinCel6A Although the entire backbones of
CcCel6C, HjeCel6A and HinCel6A are essentially
identical, the two loops of the three enzymes are not
exactly superposed (Fig 1B) In HinCel6A [7], a
mag-nesium ion forms a hexa-coordinated geometry
involved in the crystal contacts and the same
magne-sium-mediated geometry is found in the unliganded
CcCel6C, CcCel6C–pNPG3 and CcCel6C–cellobiose
Here, this magnesium ion is located close to Asp109
in loop-1 Another hexa-coordinated magnesium ion,
which is observed near Glu33, is found only in
CcCel6C–cellobiose
Ligand-bound structures Comparing the ligand-bound structures of HjeCel6A and HinCel6A with CcCel6C–pNPG3 and CcCel6C– cellobiose enabled us to label the subsites of CcCel6C
In CcCel6C–pNPG3, electron density for two ligand molecules was seen in the active site (Fig 3A) The molecule bound to subsites )3 to )1 was modelled as p-nitrophenyl b-d-cellobioside (pNPG2, not pNPG3) (average B = 25.4 A˚2; Figs 3A, 4A), and a glucose unit at the nonreducing end of pNPG3 was not identi-fied in the difference Fourier map The other molecule bound to subsites +2 to +4 was also modelled as pNPG2 (Figs 3A, 4B), but the map is better resolved
at the lower contoured level (average B = 40.8 A˚2) Weak electron density is present at subsite +1, but we could not place the models In CcCel6C–cellobiose, electron density for two ligand molecules, a cellobiose molecule bound to subsites +1 and +2 and a glucose
Table 1 Data collection and refinement statistics.
Data collection
Cell dimensions
R merge 0.025 (0.090)a 0.036 (0.184)a 0.064 (0.175)a Refinement statistics
rmsd
Number of atoms
Average B (A˚2 )
a The values for the highest resolution shells are given in parentheses.
Trang 4molecule bound to subsite )2, were identified
(Fig 3B) The cellobiose molecule gives a clear
elec-tron density map (average B = 23.0 A˚2), whereas part
of the density for the glucose molecule is not visible
(average B = 25.8 A˚2) A similar single glucose
mole-cule has been found in subsite )2 of the HinCel6A–
cellobiose complex, but it remains unclear whether the
glucose molecule is part of cellobiose or from
contami-nation in the commercial cellobiose preparation [18]
The structures of CcCel6C–pNPG3,
CcCel6C–cello-biose, HinCel6A–cellobiose [18] and
HjeCel6A–methyl-cellobiosyl-4-thio-b-cellobioside [(Glc)2-S-(Glc)2] [8]
were superposed to depict the characteristics of the
ligand binding site of CcCel6C (Fig 3C) The glucose
units in subsites )2, +1, +2 and +3 (abbreviated as
Glc )2, +1, +2 and +3, respectively) overlaid well,
whereas the aromatic ring of pNPG3 in subsite)1 was
at a position markedly different from that of Glc)1
Study of the HjeCel6A–(Glc)2-S-(Glc)2 complex has shown that Glc )1 adopts a distorted conformation, and many hydrogen bonds between HjeCel6A and Glc )1 appear to stabilize this energetically unfavoured conformation [8] The p-nitrophenyl group of pNPG3, however, is not able to form similar hydrogen bonds with CcCel6C, resulting in the different position in the active site
Although controversy exists concerning the active site residues of GH6 enzymes [19], two Asp residues are suggested to be catalytic [7] The sequence align-ment of HjeCel6A, HinCel6A and CcCel6C (Fig 2) indicated that Asp150 and Asp334 of CcCel6C are the potential catalytic residues and could act as a proton donor and a base, respectively Another aspar-tic acid residue (Asp175 of HjeCel6A) has been pro-posed to contribute to the electrostatic stabilization
of the partial positive charge in the transition state
R343 D109
D150
R343 D109
D150
A
B
C
Fig 1 Overall structures of CcCel6C.
(A) Stereoview of CcCel6C–cellobiose
shown as a ribbon model a-Helices,
b-strands and disulfide bridges are indicated
in blue, orange and green, respectively.
Cellobiose and glucose molecules bound to
the active site are shown in red Side chains
of Asp109, Asp150 and Arg343 are shown
in pink (B) Stereoview of the Ca backbone
of CcCel6C–cellobiose (red), which is
super-posed on those of HjeCel6A–(Glc)2-S-(Glc)2
(yellow; PDB id, 1QK2), HinCel6A–cellobiose
(cyan; PDB id, 2BVW) and the
endoglucan-ase HinCel6B (gray; PDB id, 1DYS) The
ligands bound to the active site are
illustrated as stick models (C) Comparison
of the Ca backbones of unliganded CcCel6C
(blue), CcCel6C–pNPG3 (green) and
CcCel6C–cellobiose (red).
Trang 5[20], and a homologous residue in CcCel6C is
proba-bly Asp102 Two distinct conformations for the
cata-lytic acid have been observed in HjeCel6A and
HinCel6A, and both are proposed to be important in
the catalysis [7,20] The Fo–Fc omit map shows that
these two conformations are present for Asp150 in
CcCel6C–cellobiose (Fig S1A), but in CcCel6C–
pNPG3, one of the conformations is not seen,
proba-bly due to steric hindrance with the p-nitrophenyl
group of pNPG3
To probe the interaction between CcCel6C and the
ligands, CcCel6C–pNPG3 and CcCel6C–cellobiose
were analysed using the program ligplot [21], and
taken together, 21 amino acid residues appear to
par-ticipate in ligand binding Table 2 lists these residues,
plus the three conserved aspartic acid residues
pro-posed to be involved in catalysis The amino acid
resi-dues in subsites)2 to +4 are highly conserved among
CcCel6C, HjeCel6A, HinCel6A and HinCel6B Four
key tryptophan residues involved in substrate stacking
interactions are fully conserved in CcCel6C as Trp61
(subsite )2), Trp297 (+1), Trp198 (+2) and Trp201
(+4) (Fig 2) as previously described [9,22] A tyrosine
residue critical for the distortion of Glc )1 (Tyr169 in HjeCel6A) [23–25] is identified in CcCel6C as Tyr86
The enclosed tunnel The cellobiohydrolases have been characterized by the enclosed tunnel as described above A conspicuous feature of CcCel6C is that the enclosed tunnel is wider than those of HjeCel6A and HinCel6A (Fig 5A–C), and loop-1 and loop-2 of CcCel6C revealed a more open structure than those of HjeCel6A and HinCel6A The conformational changes in the two loops of Hje-Cel6A and HinHje-Cel6A have been reported; binding of the ligands such as (Glc)2-S-(Glc)2 or cellobiose results
in a narrowing of the tunnel, and the additional empty space is not seen in the vicinity of the ligands [8,18] In CcCel6C, however, the two loops of the unliganded CcCel6C, CcCel6C–pNPG3 and CcCel6C–cellobiose are superposed well (Fig 1C), and the rmsd values for all backbone atoms are 0.109 A˚ (between unliganded CcCel6C and CcCel6C–pNPG3) and 0.159 A˚ (between unliganded CcCel6C and CcCel6C–cellobiose) The possibility that the two loops are trapped in the open
Fig 2 Comparison of amino acid sequences of CcCel6C and related enzymes The sequences were aligned using the CLUSTALW2 server, and manual adjustment was carried out based on the comparison of the crystal structures The numbering of amino acid residues and secondary structures (a1–a8and b0–bVII) are given Residues listed in Table 2 are printed with a red ⁄ pink (subsites )3 to )1) or blue ⁄ cyan (subsites +1 to +4) background The two loops, loop-1 and loop-2, are under-lined Other symbols: arrow, Asp150 and Arg343; asterisk, three conserved aspartic acid and four conserved tryptophan described in the text; dashed line, disulfide bridge.
Trang 6conformations by crystal packing could not be
excluded, as the crystals of the complex structures were
obtained by soaking with pNPG3 or cellobiose
In HjeCel6A–(Glc)2-S-(Glc)2, however, the
conforma-tional changes in the tunnel-forming loops have been
observed by addition of the ligand after the crystals
had reached full size [8] The most significant
move-ment of HinCel6A occurs at residues Ala183 to
Gly188 [18], and the sequence of HjeCel6A⁄ HinCel6A,
A-L⁄ A-A-S-N-G, is composed of amino acids with
rel-atively small side chains The corresponding region of
CcCel6C (residues 105–110) is A-K-A-S-D-G, which
contains a bulky lysine residue It appears that the
conformational change in the two loops of CcCel6C is
less favourable and the tunnel still has an open space
near the binding sites of cellobiose or pNPG3
Loop-1 contacts with loop-2 mainly via an
interac-tion between Asp109 and Arg343 In unliganded
CcCel6C, the electron density map for Asp109 is clear,
but the 2Fo–Fc map for Arg343 is better resolved at
the lower contoured level of 0.8r, and two hydrogen
bonds, Asp109 OD2-Arg343 NE and Ser108 O-Arg343
NH1 could form directly between loop-1 and loop-2 in
this model As for Asp109 in both CcCel6C–pNPG3
and CcCel6C–cellobiose and Arg343 in CcCel6C–
pNPG3, the Fo–Fc omit maps show that there are at least two different conformations (Fig S1B) This observation suggests that the enclosed tunnel of CcCel6C is not completely ‘enclosed’, although the ligand molecules are unable to pass through the open-ing between loop-1 and loop-2
From subsites )2 to +4, only one serine residue, Ser236, is not conserved in the other fungal cellobiohy-drolases (Table 2), and the corresponding residue of HjeCel6A and HinCel6A is alanine (Ala304 and Ala309, respectively) As described in the previous section, the tunnel-forming loops of HinCel6A are changed to adopt the closed conformation when the ligands bind to the active site As a result, in HinCel6A–cellobiose, a serine residue in loop-1,
A
B
Fig 4 Schematic drawing of the amino acid residues interacting with the ligands observed at subsites )3 to )1 (A) and +2 to +4 (B) Symbols: open circle, oxygen atom; closed circle, carbon atom; gray circle, nitrogen atom; dashed line, hydrogen bond The residues involved in hydrophobic interactions are illustrated.
K30
S26
D334
Y96 D102 D150 W198
A
B
C
Fig 3 Comparison of the ligands bound to the active site (A) The
pNPG3 F o –F c electron density maps at the 2.0 r contoured level.
The subsite numbers are labelled from )3 to +4 (B) The cellobiose
Fo–Fcelectron density maps at the 2.0 r contoured level (C)
Over-lays of the ligands in CcCel6C–pNPG3 (green), CcCel6C–cellobiose
(red), HjeCel6A–(Glc) 2 -S-(Glc) 2 (yellow; PDB id, 1QJW) and
Hin-Cel6A–cellobiose (cyan; PDB id, 2BVW) Some critical residues
described in the text are indicated in black.
Trang 7Ser186, can directly form hydrogen bonds with the
ligand [18] The conformational change of HjeCel6A–
(Glc)2-S-(Glc)2 has been reported to be more
compli-cated, and four states (most closed, more open, even more open, and most open) of the loop have been identified The complex of wild-type HjeCel6A–(Glc)2 -S-(Glc)2 (PDB id, 1QK2) has been observed in the
‘more open’ form and the corresponding serine residue, Ser181, does not interact with (Glc)2-S-(Glc)2 The Y169F mutant of HjeCel6A complexed with (Glc)2 -S-(Glc)2(PDB id, 1QJW), on the other hand, adopts the
‘most closed’ form, and Ser181 is pointed into the )1 site and OG atom of the serine residue hydrogen bonds with O5 of Glc )1, O4 of Glc )1, and O2 of Glc )2 [8] It is not easy to interpret the role of the Ser181⁄ 186 residue during the catalysis, but they appear to stabilize the distorted conformation of Glc )1 However, the significant conformational changes
of the two loops of CcCel6C were not observed (Fig 1C) and in CcCel6C–cellobiose, Ser108, the posi-tion equivalent to Ser181⁄ Ser186, does not directly hydrogen bond with the ligand In the endoglucanases, the corresponding residue of Ser236 is found to be ser-ine (Ser221, HinCel6B; Ser189, Thermobifida fusca Cel6A) and in the complex of Thermobifida fusca, Cel6A with (Glc)2-S-(Glc)2, Ser189 hydrogen bonds with O6 of the distorted glucose unit Glc )1 [25] The role of Ser236 in CcCel6C is probably similar to that
of Ser189 in Thermobifida fusca Cel6A
Subsite )3
In contrast to the high similarity of subsites from )2
to +4, two amino acid residues involved in subsite )3
of CcCel6C, Ser26 and Lys30, are strikingly differ-ent from those of HjeCel6A (Tyr103 and Glu107, respectively) and HinCel6A (Tyr104 and Glu108,
Table 2 Amino acid residues interacting with the ligands or
poten-tially involved in the catalysis, and the corresponding residues of
HjeCel6A, HinCel6A and HinCel6B pNP, p-nitrophenyl group.
Closest
Glc ⁄ pNP a CcCel6C HjeCel6A HinCel6A HinCel6B
a The closest Glc ⁄ pNP is determined based on the cartoon
gener-ated using the program LIGPLOT b Amino acid residues that are not
identical to those of CcCel6C are given in parentheses.
Fig 5 Surface models of CcCel6C and related enzymes (A–C) Overall structures of CcCel6C (A), HinCel6A (B) and the endoglu-canase HinCel6B (C) (D–F) Close-up views
in the vicinity of subsite )3 of CcCel6C (D), HinCel6A (E) and HinCel6B (F) To generate the models, the structure of
CcCel6C–pNPG3 was superposed on those
of HinCel6A (PDB id, 2BVW) and HinCel6B (PDB id, 1DYS), and pNPG2 was placed on the models Glc )3 is labeled as )3 Residues forming protruding knobs at the entrance of the cleft are shown in yellow and red, and the distances between them (A ˚ ) are indicated Ser26 and Lys30 of CcCel6C and the corresponding residues are indicated in cyan and pink, respectively.
Trang 8respectively) For the cellobiohydrolases, subsite )3 is
typically presumed unnecessary to produce cellobiose,
but the studies of HinCel6A have revealed no
substan-tial evidence to completely negate a)3 subsite [18] and
subsites from)4 to +4 of HinCel6A are proposed [9]
In CcCel6C, Glc)3 of pNPG3 not only forms
mul-tiple hydrogen bonds with Ser26, Lys30 and Trp61
through water molecules, but also makes hydrophobic
contacts with Pro329, Glu332 and Gly361 (Fig 5D)
However, modelling pNPG3 in its CcCel6C-bound
conformation with either HjeCel6A or HinCel6A, Glc
)3 causes steric conflict with a tyrosine residue
(HjeCel6A Tyr103 and HinCel6A Tyr104) (Fig 5E)
The cleft for subsite)3 of CcCel6C is also apparently
wider than those of HjeCel6A or HinCel6A (Fig 5A,
B) Asp64 and Glu362 of CcCel6C form protruding
knobs at the entrance of the cleft, and the distance
between atom OD2 of Asp64 and atom OE2 of
Glu362 is 9.8 A˚ (Fig 5D) Similar knobs, which are
formed by Arg140 and Gln433, are observed at the
entrance of the cleft of HinCel6A, but the distance
between atom NH1 of Arg140 and atom NE2 of
Gln433 is only 6.9 A˚ (Fig 5E) These observations
indicate that the accessibility of subsite)3 of CcCel6C
is less restricted than those of HjeCel6A and
Hin-Cel6A The cleft for subsite )3 of CcCel6C is rather
similar to that of the endoglucanase HinCel6B
(Fig 5C, F) The residue of HinCel6B equivalent to
Ser26 of CcCel6C is identified as Asp16, an amino acid
residue with a relatively small side chain and, thus, no
steric conflict is found if the similar placement is tested
for the endoglucanase HinCel6B The width of the
entrance of the cleft for subsite)3 (ND2 of Asn55-SD
of Met329) is 11.6 A˚, which is similar to that of
CcCel6C (Fig 5F)
Implications for enzymatic activity
The structure of CcCel6C contains the enclosed tunnel
around its active site, indicating that the enzyme has
cellobiohydrolase activity Indeed, our previous study
showed that CcCel6C hydrolysed phosphoric
acid-swollen cellulose with the release of cellobiose as a
main product [12] However, the enzyme lacks a
cellu-lose binding domain, which is necessary to hydrolyse
crystalline cellulose, and most GH6 cellobiohydrolases
have this domain In addition, we have reported that
the transcript level of CcCel6C was very low at the
active growth stage in the cellulose-degrading culture,
and almost the same transcript level was detected at
the active growth stage in the glucose culture The
transcript level also did not change when the mycelia
were transferred to a medium containing glucose,
cellobiose or no carbon source [11] These findings sug-gest that the physiological role of CcCel6C does not involve the degradation of crystalline cellulose
Cellulose is insoluble in water; for the enzyme to recognize it, the insoluble cellulose must be converted into soluble saccharides, such as cellobiose and cellool-igosaccharides In the past several decades, it has been assumed that low and constitutive levels of cellulases react with cellulose to produce a soluble molecule that enters the cell and induces transcription of cellulase genes [26,27] Considering the results of our biochemi-cal and transcript analyses, CcCel6C probably pro-duces a small amount of cellobiose when cellulose is present Similar activity has been reported in a GH7 enzyme produced by basidiomycete
Phanerochae-te chrysosporium, PcCel7A [28–30] This enzyme shows the amino acid sequence corresponding to an active site tunnel also shown in GH7 cellobiohydrolases, but like CcCel6C, lacks a cellulose binding domain A low level of PcCel7A transcripts was observed in the cul-ture containing cellulose, whereas the transcripts were detected in glucose culture In a homology modelling analysis, the enzyme was expected to have endo-type activity [31] These enzymatic and transcriptional prop-erties are very similar to those of CcCel6C and, thus, PcCel7A might also produce an inducer of cellulase genes Recently, it was also reported that basidiomy-cete Coniophora puteana has GH6 and GH7 enzymes without a cellulose binding domain [32] Therefore, the existence of cellobiohydrolases lacking a cellulose binding domain might characterize soluble cellulose degradation specifically in basidiomycetes because this type of enzyme was not found in ascomycetes Many reports have shown that cellobiohydrolases are typically active on crystalline cellulose [1] The fungal cellobiohydrolases, HjeCel6A and HinCel6A, have the cellulose binding domain [33,34] Specific conforma-tional changes have been observed in the two active site enclosing loops of HjeCel6A and HinCel6A that seem to be critical in hydrolysing the crystalline cellulose [8,18] CcCel6A, whose amino acid sequence
is highly similar to those of HjeCel6A and HinCel6A, probably has similar activity on amorphous cellulose
in Coprinopsis cinerea
The structures determined here indicate that CcCel6C has an enclosed tunnel similar to that of HjeCel6A and HinCel6A The tunnel is, however, wider and more open than these fungal cellobiohydro-lases, and virtually no conformational change in the two loops of CcCel6C is induced The ligand binding cleft of CcCel6C is also wider due to the absence of the bulky tyrosine residue in subsite )3 (Fig 5), and the structures of subsites)1 and )3 of CcCel6C
Trang 9resem-ble those of the endoglucanases HinCel6B rather than
HjeCel6A and HinCel6A We have reported that
CcCel6C hydrolyses the chemically modified cellulose
derivative, carboxymethyl cellulose, whereas CcCel6A
does not [12] The open tunnel and the wide cleft are
probably suitable for the hydrolysis of carboxymethyl
cellulose Carboxymethyl cellulose and amorphous
cellulose have been reported to be substrates of most
endoglucanases, indicating that the enzyme activity is
mainly directed towards amorphous regions in the
cellulose molecule [1] The results described above lead
us to conclude that the architecture of CcCel6C could
be suitable for hydrolysing amorphous cellulose to
serve cellobiose, an inducer for the expression of
CcCel6A in Coprinopsis cinerea
Experimental procedures
Enzyme preparation and crystallization
The expression and purification of CcCel6C were carried out
as described previously [35] Briefly, recombinant CcCel6C
fused with a His-tag was produced in Escherichia coli
BL21(DE3) cells and purified with Ni-NTA agarose (Qiagen,
Hilden, Germany) The enzyme was crystallized at 20C
using the hanging drop vapour diffusion method, where
1 lL CcCel6C (21.5 mgÆmL)1) was mixed with the same
vol-ume of well solution (100 mm Hepes⁄ KOH pH 7.0, 30%
polyethylene glycol 8000, 150 mm magnesium acetate) The
obtained crystal was transferred to a cryo-solution of 40%
(w⁄ v) polyethylene glycol 8000 in well solution and flash
fro-zen in a stream of nitrogen gas The crystal of the complex
of pNPG3 (Seikagaku Corporation, Tokyo, Japan) or
cello-biose was obtained by soaking in the same well solution
(100 mm Hepes⁄ KOH pH 7.0, 30% polyethylene glycol
8000, 150 mm magnesium acetate) containing 60 mm
pNPG3 for 2 h or 220 mm cellobiose for 5 min, and the
solution containing the ligand also acted as a cryoprotectant
The diffraction data were collected at beamline PF-AR
NW12 (Photon Factory, Tsukuba, Japan) The data were
processed and scaled with the program hkl2000 [36]
(Table 1)
Model building and structure refinement
The structure of CcCel6C was solved by molecular
replace-ment with the program molrep in the ccp4 suite [17], and
a model of HinCel6A (PDB id, 1BVW) [7] was employed
as a probe model The automated model building was
per-formed with the program arp⁄ warp [37] The refinement
was carried out using the program refmac in the ccp4
suite, and anisotropic refinement was applied for data
bet-ter than 1.2 A˚ resolution Manual adjustment and
rebuild-ing of the model were carried out with the program coot
[38], and the models for the ligands were built from both 2Fo–Fcand Fo–Fcelectron density maps Solvent molecules were introduced using the program arp⁄ warp Refinement statistics are listed in Table 1 Superpositioning of CcCel6C with other protein structures and calculation of the rmsd values were carried out using the program superpose in the ccp4 suite Sequence identities were calculated using the program clustalw2 on the ebi server (http://www.ebi.a-c.uk/Tools/clustalw2/) [39] with the default values Figures were generated using ligplot [21] and pymol (http:// www.pymol.org/) The coordinates and structure factors of unliganded CcCel6C, CcCel6C–pNPG3 and CcCel6C–cello-biose have been deposited in the Protein Data Bank under the accession codes 3A64, 3ABX and 3A9B, respectively
Acknowledgements
This research was supported, in part, by the Green Biomass Research for Improvement of Local Energy Self-sufficiency Program of the Ministry of Education, Culture, Sports, Science and Technology of Japan This research was performed with the approval of the Photon Factory Advisory Committee (2008G013), the National Laboratory for High Energy Physics, Tsukuba, Japan
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