Báo cáo khoa học: The crystal structure of a xyloglucan-specific endo-b-1,4glucanase from Geotrichum sp. M128 xyloglucanase reveals a key amino acid residue for substrate specificity potx

The crystal structure of a xyloglucan-specific endo-
b
-1,4-
glucanase from Geotrichum sp. M128 xyloglucanase
reveals a key amino acid residue for substrate specificity
Katsuro Yaoi
1,
*, Hidemasa Kondo
2,
*, Ayako Hiyoshi
1
, Natsuko Noro
2
, Hiroshi Sugimoto
3
,
Sakae Tsuda
2,4
and Kentaro Miyazaki
1,5
1 Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki,
Japan
2 Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), Toyohira,
Sapporo, Hokkaido, Japan
3 Riken SPring-8 Center, Harima Institute, Hyogo, Japan
4 Division of Biological Science, Graduate School of Science, Hokkaido University, Sapporo, Japan
5 Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tsukuba, Ibaraki, Japan
Introduction
Xyloglucan is a major hemicellulose in the primary cell
wall of plants, where it associates with cellulose micro-
fibrils via hydrogen bonds to form a cellulose–xyloglu-

can network. Xyloglucan consists of a cellulose-like
main chain of b-1,4-glucan, which is frequently
branched with xylose side chains to form a-d-Xylp-
Keywords
endo-b-1,4-glucanase; glycoside hydrolase
family 74; xyloglucan; xyloglucanase;
b-1,4-glucan
Correspondence
K. Yaoi, Institute for Biological Resources
and Functions, National Institute of
Advanced Industrial Science and Technology
(AIST), Tsukuba Central 6, 1-1-1 Higashi,
Tsukuba, Ibaraki 305-8566, Japan
Fax: +81 29 861 6733
Tel: +81 29 861 6065
E-mail:
*These authors contributed equally to this
study
Database
The coordinates of the structure for XEG
have been deposited in the Protein Data
Bank under the accession number 3A0F
(Received 14 May 2009, revised 3 July
2009, accepted 8 July 2009)
doi:10.1111/j.1742-4658.2009.07205.x
Geotrichum sp. M128 possesses two xyloglucan-specific glycoside hydrolases
belonging to family 74, xyloglucan-specific endo-b-1,4-glucanase (XEG)
and oligoxyloglucan reducing-end-specific cellobiohydrolase (OXG-RCBH).
Despite their similar amino acid sequences (48% identity), their modes of
action and substrate specificities are distinct. XEG catalyzes the hydrolysis

of xyloglucan polysaccharides in endo mode, while OXG-RCBH acts on
xyloglucan oligosaccharides at the reducing end in exo mode. Here, we
determined the crystal structure of XEG at 2.5 A
˚
resolution, and compared
it to a previously determined structure of OXG-RCBH. For the most part,
the amino acid residues that interact with substrate are conserved between
the two enzymes. However, there are notable differences at subsite posi-
tions )1 and +2. OXG-RCBH has a loop around the +2 site that blocks
one end of the active site cleft, which accounts for its exo mode of action.
In contrast, XEG lacks a corresponding loop at this site, thereby allowing
binding to the middle of the main chain of the substrate. At the )1 site in
OXG-RCBH, Asn488 interacts with the xylose side chain of the substrate,
whereas the )1 site is occupied by Tyr457 in XEG. To confirm the contri-
bution of this residue to substrate specificity, Tyr457 was substituted by
Gly in XEG. The wild-type XEG cleaved the oligoxyloglucan at a specific
site; the Y457G variant cleaved the same substrate, but at various sites.
Together, the absence of a loop in the cleft and the presence of bulky
Tyr457 determine the substrate specificity of XEG.
Abbreviations
GH74, glycoside hydrolase family 74; Glc, glucose; OXG-RCBH, oligoxyloglucan reducing-end-specific cellobiohydrolase; XEG, xyloglucan-
specific endo-b-1,4-glucanase; Xyl, xylose.
5094 FEBS Journal 276 (2009) 5094–5100 ª 2009 The Authors Journal compilation ª 2009 FEBS
(1 fi 6)-b-d-Glcp. Other sugars such as galactose,
arabinose and fucose may also be present on the side
chains in various branching patterns, depending on the
plant species. Structural studies on xyloglucans suggest
that most consist of repeating units of either XXXG
(XXXG-type) or XXGG (XXGG-type) [1], where G
refers to an unbranched Glc residue and X represents

the xylose (Xyl)-branched Glc a-d-Xyl p-(1 fi 6)-b-d-
Glcp [2]. Thus, XXXG-type xyloglucans have three
consecutive backbone residues with Xyl side chains
and a fourth unbranched Glc residue, and XXGG-type
xyloglucans have two consecutive branched backbone
residues and two unbranched backbone residues.
Various microorganisms that degrade the plant cell
wall produce endo-b-1,4-glucanases that hydrolyze
xyloglucan. Most of them cleave the glycosidic bond
of the unbranched Glc residues in the backbone chain.
Typically, treatment of an XXXG-type xyloglucan
with these endoglucanases generates oligosaccharides
with a tetrasaccharide backbone (XXXG). For many
years, endo-b-1,4-glucanases have been considered to
be a subgroup of cellulases (EC 3.2.1.4). Recently,
however, it has been clarified that some endo-b-1,4-
glucanases display high activity toward xyloglucan but
have limited or no activity against cellulose. These
xyloglucan-specific endo-b-1,4-glucanases have been
classified as a new enzyme family (EC 3.2.1.151), desig-
nated xyloglucan-specific endo-b-1,4-glucanases (XEG)
or xyloglucanases. Many xyloglucanases belonging to
glycoside hydrolase families 5, 12, 44 and 74 (http://
www.cazy.org/) have now been identified. Of these,
glycoside hydrolase family 74 (GH74) enzymes exhibit
high specificity towards xyloglucan [3–9].
Previously, we have reported the cloning, purifica-
tion and characterization of two GH74 enzymes, oligo-
xyloglucan reducing-end-specific cellobiohydrolase
(OXG-RCBH) [6] and xyloglucan-specific endo-b-1,4-

glucanases (XEG) [7], from Geotrichum sp. M128.
OXG-RCBH (EC3.2.1.150) is a unique exo-type
enzyme that recognizes the reducing end of xyloglucan
oligosaccharides. Its substrate recognition mechanism
has been investigated using various oligosaccharide
substrates [6], and mutational and detailed structural
studies have revealed its unique mechanism [10,11].
The exo activity of OXG-RCBH is based on a loop at
one side of the active site cleft. In addition, residue
Asn488 in the active site cleft recognizes the Xyl side
chain at the )1 site, conferring the unique substrate
specificity. On the other hand, XEG showed typical
endo activity [12]. It hydrolyzes xyloglucan polymers
randomly. In addition, XEG catalyzes hydrolysis of
the glycosidic bond of unbranched Glc residues, sug-
gesting a difference between the two enzymes in the
three-dimensional structures of the active sites. In this
study, we determined the crystal structure of XEG in
order to determine the structural basis for its substrate
specificity.
Results and discussion
Overall structure of XEG
Crystals were grown using 0.1 m MES buffer, pH 5.8–
6.0, and 3–6% w ⁄ v PEG 8000. They were elongated
rod- or needle-shaped crystals, belonging to the P3
2
21
trigonal space group, with unit-cell parameters of
a = b = 135.2 A
˚

and c = 119.9 A
˚
. One protein
molecule of XEG existed in an asymmetric unit of the
crystal. X-ray diffraction data were collected at 2.5 A
˚
resolution, with an R
merge
of 0.096 and 98.4% com-
pleteness. The structure was determined using the
molecular replacement method and was refined against
20–2.5 A
˚
intensity data. The crystallographic R factor
and free R factor were 0.236 and 0.276, respectively.
Table 1 summarizes the statistics of X-ray data collec-
tion, and the results for the structural refinement of
Table 1. Statistics for data collection using the Beamline BL44B2
at SPring-8 and structure refinement of XEG.
Parameter Value
Data collection
Wavelength (A
˚
) 1.0000
Resolution
a
(A
˚
) 20–2.5 (2.59–2.5)
R

merge
a,b
0.096 (0.199)
Observed reflections 267 842
Independent reflections 43 425
Completeness
a
(%) 98.4 (93.2)
Multiplicity
a
6.2 (6.0)
<I ⁄ r(I)>
a
17.9 (8.6)
Refinements
Resolution range
a
(A
˚
) 20–2.5 (2.56–2.5)
R factor
a,c
0.236 (0.251)
Free R factor
a,c
0.276 (0.296)
Number of non-hydrogen atoms
Protein 5 676
Water 107
Root mean square deviations from

ideality (bond length) (A
˚
)
0.015
Root mean square deviations from
ideality (angle) (°)
1.64
a
Numbers in parentheses are values for the highest-resolution
shell: 2.59–2.5 A
˚
for the data collection and 2.56–2.5 A
˚
for the
refinement.
b
R
merge
¼
P
h
P
j
<Iðh Þ> À I
j
ðh Þ





=
P
h
P
j
I
j
ðhÞ, where
<I(h)> is the mean intensity of a set of equivalent reflections.
c
R factor ¼
P
h
F
obs
ðhÞÀF
calc
ðhÞ
jj
=
P
h
F
obs
ðh Þ
jj
, where F
obs
and F
calc

are the observed and calculated structural factors, respectively.
K. Yaoi et al. Key amino acid residue of Geotrichum XEG
FEBS Journal 276 (2009) 5094–5100 ª 2009 The Authors Journal compilation ª 2009 FEBS 5095
XEG. Figure 1 shows the electron density map of
XEG for the region corresponding to the exo-loop of
OXG-RCBH. The coordinates and structure factors
have been deposited in the Protein Data Bank (PDB)
(accession code 3A0F).
The structure of XEG was compared with those of
other GH74 xyloglucan-specific enzymes, OXG-RCBH
and Clostridium Xgh74A. The overall structures of
XEG (this study), OXG-RCBH in complex with
substrate XXXG (PDB code 2EBS) and Xgh74A in
complex with substrates XLLG and XXLG (L refers to
a b-d-Galp-(1 fi 2)- a-d-Xylp-(1 fi 6)-b-d-Glcp) (PDB
code 2CN3) [13] are illustrated in Fig. 2. All enzymes
have two structurally similar domains, each consisting
of a seven-bladed b-propeller. These are located
tandemly in the N- and C-terminal halves of the poly-
peptide, and are joined on each edge of the domain to
form a bivalve-like shape. XEG can be superimposed
onto OXG-RCBH with an RMSD of 1.07 A
˚
by the
corresponding 683 Ca atoms among 756 residues, indi-
cating close similarities between their domain structures
as well as the relative positions of the two domains.
The active site cleft is located between the N- and
C-domains. One apparent difference between XEG
and OXG-RCBH is at the active site. In OXG-RCBH,

a loop comprising 11 amino acid residues protrudes
from the N-domain, blocking one end of the cleft.
However, XEG lacks a corresponding loop because of
deletion of those residues. The loop structure is
responsible for the exo-activity of OXG-RCBH, and
the absence of the loop is associated with endo-activity
in XEG. Therefore, it is most likely that the endo-
activity of XEG is primarily attributable to the active
site cleft being open at both ends. In the case of
Xgh74A, the active cleft is open. Although a precise anal-
ysis of the mode of action of Xgh74A has not been
performed, Xgh74A appears to be an endoglucanase
because it has an open cleft.
Comparison of the XEG and OXG-RCBH
active sites
Figure 3 shows a close-up view of the active sites of
XEG and OXG-RCBH. The main chain of XEG
exhibits close similarity to that of OXG-RCBH, and
the side chain conformations involved in substrate
interactions are very well conserved, with some excep-
tions (see below for details). Previously, we have iden-
tified the catalytic residues Asp35 (base) and Asp465
(acid) in OXG-RCBH [10]. Equivalent residues were
identified in the active site of XEG (Asp34 and
Asp458). Thus, it is conceivable that XEG and
OXG-RCBH recognize the backbone of the b-1,4-glu-
can in a similar manner, despite their differences in
substrate specificity and mode of hydrolysis.
Two notable differences, located at the )1 and +2
sites, were observed. OXG-RCBH functions in exo

mode owing to its unique loop structure around the
+2 site at one end of the cleft (Figs 2B and 3, shown
in red). The Xyl side chain at the +2 site inhibits
enzymatic activity due to steric hindrance between the
side chain and the exo-loop. Previously, we demon-
strated that deletion of the loop region leads to loss of
specificity, as the resultant enzyme could catalyze
cleavage at various sites of the substrate by endo activ-
ity [10]. XEG does not have a corresponding loop;
both sides of the cleft are open. This result, and those
of the previous experiment with loop-deleted OXG-
RCBH [10], strongly suggest that the basis for the endo
activity of XEG is the absence of the exo loop.
The second difference was observed at the )1 site.
In OXG-RCBH, Asn488 interacts with a Xyl side
chain at the )1 site. Previously, we have shown that
recognition of the Xyl side chain at the )1 site is a key
determinant for the substrate specificity of OXG-
RCBH [6]. However, the corresponding position in
XEG is occupied by the bulky side chain of Tyr457,
which appears to hinder binding of the Xyl side chain
at this position. This may explain why XEG prefers
Fig. 1. Stereoview of the r
A
-weighted 2m|F
obs
| ) D|F
calc
| map of
XEG at 2.5 A

˚
resolution, contoured at 1.5r. The map shows the
vicinity of the region corresponding to the exo loop, which is pres-
ent in RCBH and absent in XEG. The XEG molecule is also shown
as a stick model. The exo loop of RCBH is drawn as a trace of Ca
atoms in orange. The image was produced using the program
CCP4MG [22].
Key amino acid residue of Geotrichum XEG K. Yaoi et al.
5096 FEBS Journal 276 (2009) 5094–5100 ª 2009 The Authors Journal compilation ª 2009 FEBS
substrates with an unbranched Glc residue at the )1
site and catalyzes cleavage of the glycosidic bond of
unbranched Glc residues.
Activity of the XEG mutant Y457G
To confirm the role of Tyr457, we constructed a XEG
variant, Y457G, which was expressed in Escherichia
coli, purified, and characterized. The kinetic constants
of Y457G against tamarind seed xyloglucan were
determined. The apparent K
m
values of the wild-type
and Y457G enzymes were 0.47 and 0.43 mgÆmL
)1
,
respectively, and their specific activities were 15.7 and
3.97 unitÆmg
)1
protein, respectively. Next, the substrate
specificity was investigated using a tetradecasaccharide,
XXXGXXXG. Wild-type XEG generated only XXXG
(Fig. 4), because of its strict specificity for glycosidic

bonds of the unbranched Glc residue. By contrast, the
Fig. 2. Schematic drawing of the entire
structures (traces of Ca atoms) of XEG (A),
the OXG-RCBH ⁄ substrate (XXXG) complex
(B) and the Xgh74A ⁄ substrate (XLLG and
XXLG) complex (C). The substrates are
shown as stick models. The exo loop of
OXG-RCBH (Gly375–His385) is shown
in red.
K. Yaoi et al. Key amino acid residue of Geotrichum XEG
FEBS Journal 276 (2009) 5094–5100 ª 2009 The Authors Journal compilation ª 2009 FEBS 5097
Y457G mutant released various oligosaccharides from
cleavage of XXXGXXXG. The structures of these
products were X, XG, GX, XX, XXG, XXX and
XXXG, as confirmed by MALDI-TOF MS combined
with enzymatic treatments of the reaction products
using isoprimeverose-producing oligoxyloglucan hydro-
lase from Oerskovia [14] and b-glucosidase from
almond, Prunus dulcis, as described previously [8]
(data not shown). On the basis of the product patterns,
we propose the possible cleavage sites shown in Fig. 4.
Therefore, the Y457G variant can catalyze cleavage of
the glycosidic bond of branched Glc residues, i.e. the
Glc residue at the )1 site can be branched. The
Y457G mutant enzyme was less selective, indicating
that Y457 plays an important role in determining sub-
strate specificity.
Conclusion
Geotrichum M128 produces two GH74 enzymes, XEG
and OXG-RCBH. They share 48% amino acid identity

and three-dimensional structures. The majority of the
residues interacting with the substrate are well-con-
served. However, the residues that determine substrate
specificity and mode of action were distinct. XEG and
OXG-RCBH display endo and exo modes of action,
respectively. A loop structure that determines the exo
action of OXG-RCBH is not present in XEG.
Fig. 3. Comparison of the active site from )2 to +2 for XEG and the OXG-RCBH ⁄ XXXG complex. The stereo views of XEG and OXG-RCBH
are shown in blue and purple, respectively. The substrate XXXG and the amino acids that interact with the substrate are shown as stick
models. The substrate is colored blue, light green, green and orange at the )2, )1, +1 and +2 sites, respectively. The exo loop of OXG-
RCBH is colored in red.
Fig. 4. HPLC analysis of the digestion prod-
ucts of the xyloglucan oligosaccharide
XXXGXXXG by wild-type XEG and the
Y457G mutant. The xyloglucan oligosaccha-
ride (XXXGXXXG) was incubated with wild-
type XEG or the Y457G mutant, and the
products were analyzed by HPLC. The pro-
posed cleavage sites are indicated by
arrows. (A) Oligosaccharide marker. (B)
Products from cleavage by wild-type XEG.
(C) Products from cleavage by the Y457G
mutant.
Key amino acid residue of Geotrichum XEG K. Yaoi et al.
5098 FEBS Journal 276 (2009) 5094–5100 ª 2009 The Authors Journal compilation ª 2009 FEBS
A further difference in the active site residues
involves recognition of a Xyl side chain at the
)1 site. OXG-RCBH recognizes a Xyl side chain at
the )1 site (Asn488), whereas XEG does not. In
XEG, residue Tyr457, which corresponds to Gly464

of OXG-RCBH, appears to protrude into the )1 site,
providing a basis for the substrate specificity of XEG.
In fact, a single amino acid substitution of Tyr457 by
Gly caused a significant change in substrate recogni-
tion by XEG. These results suggest that the substrate
specificities of XEG and OXG-RCBH depend on a
limited number of residues in the substrate binding
cleft.
Experimental procedures
Purification of XEG
The gene encoding the mature region of XEG [7] was subcl-
oned into pET14-b (Novagen, Madison, WI, USA) using
NdeI and BamHI sites, resulting in fusion of a His
6
tag to
the N-terminus. The resultant product was transformed
into E. coli BL21-CodonPlus (DE3) RP (Stratagene, La
Jolla, CA, USA). Expression was induced by addition of
isopropyl-b-d-thio-galactopyranoside (final concentration
0.1 mm) for 16 h at 20 °C. The soluble, intracellular recom-
binant protein was extracted using BugBuster protein
extraction reagent (Novagen), and was purified using a
HiTrapÔ chelating column (GE Healthcare, Little Chal-
font, UK). Solid ammonium sulfate was added to the active
fractions from the column to a final concentration of
0.75 m, and the fractions were loaded onto a Resource
PHE hydrophobic interaction chromatography column
(Amersham Biosciences) equilibrated with 25 mm imidaz-
ole ⁄ HCl buffer (pH 7.4) containing 0.75 m (NH
4

)
2
SO
4
.
Bound proteins were eluted using a linear gradient of
0.75–0 m (NH
4
)
2
SO
4
in 25 mm imidazole ⁄ HCl buffer
(pH 7.4). Finally, XEG was resolved on a HiLoad 16 ⁄ 60
Superdex 200 pg column (Amersham Biosciences) in 25 mm
imidazole ⁄ HCl buffer (pH 7.4). Before crystallization,
purified XEG was concentrated to 8 mgÆmL
)1
using an
Ultrafree-15 centrifugal filter device (Millipore, Bedford,
MA, USA).
Crystal structure determination of XEG
Crystallization of XEG was performed at 20 °C using the
hanging-drop vapor-diffusion method [15]. The crystalliza-
tion conditions were initially screened using the screens
Index and PEG ⁄ Ion (both Hampton Research, Aliso Viejo,
CA), and Wizard I, Cryo I and Cryo II (all DeCODE
Genetics, Reykjavik, Iceland), and were refined by varying
the pH of the buffer and the concentration of the precipi-
tant. Prior to data collection, a crystal was transferred into

Paratone-N (Hampton Research) and mounted on a
CryoLoop (Hampton Research) of 20 lm diameter. The
mounted crystal was immersed in liquid nitrogen. Diffrac-
tion data were collected at 100 K on Beamline BL44B2 at
SPring-8 (Harima Institute, Hyogo, Japan) using an ADSC
Quantum 210 CCD detector (Area Detector Systems
Corporation, Poway, CA, USA) with 1.0000 A
˚
radiation,
and were processed using HKL2000 [16] and the ccp
4 pro-
gram suite [17]. The structure of XEG was solved by
means of the molecular replacement method, using the
AMORE program [18] in the ccp
4 package. The coor-
dinates of OXG-RCBH (PDB code 1sqj) were used as
a search model. The cns program [19] was used for
refinement against the 20–2.5 A
˚
intensity data. A ran-
domly selected portion of the diffraction data (5.0%)
was used to calculate the free R factor [20]. The pro-
gram coot [21] was used to display and correct the
structure. Figures were created using pymol (DeLano
Scientific LLC, Palo Alto, CA; http://www.
pymol.org). The coordinates were deposited in the Pro-
tein Data Bank (3A0F).
Construction of the Y457G mutant
The Y457G mutant was constructed using the QuikChangeÒ
procedure (Stratagene) with primers 5¢-TCTTCAGCGGC

ATGGGCGACCTCGGCGGCAT-3¢ and 5¢-ATGCCGC
CGAGGTCGCCCATGCCGCTGAAGA-3¢. The recombi-
nant protein was purified using a HiTrapÔ chelating
column and Resource PHE column (both Amersham
Biosciences).
Analysis of kinetic parameters
The kinetic parameters were determined at various concen-
trations of the substrate, tamarind seed xyloglucan (Dainip-
pon Sumitomo Pharma, Osaka, Japan), in 50 mm sodium
acetate buffer (pH 5.5) at 45 °C for 15 min. The bicinchoni-
nate assay was used to quantify reducing sugars. The
Michaelis constant (K
m
) and specific activity were calcu-
lated from a plot of initial reaction rates versus substrate
concentration using Prism (GraphPad Software, San Diego,
CA, USA). One unit was defined as the amount of enzyme
that released 1 lmol of glucose equivalent as reducing sug-
ars from xyloglucan per minute.
Analysis of substrate specificity
Substrate specificity was analyzed using xyloglucan tetra-
decasaccharide, XXXGXXXG, prepared as described previ-
ously [8]. The oligosaccharide (0.2 mg) was incubated with
each enzyme in 20 lLof50mm sodium acetate buffer
(pH 5.5) at 45 °C for 16 h. The resulting products were
analyzed by HPLC using an Amide-80 normal-phase
K. Yaoi et al. Key amino acid residue of Geotrichum XEG
FEBS Journal 276 (2009) 5094–5100 ª 2009 The Authors Journal compilation ª 2009 FEBS 5099
column (4.6 mm I.D. · 250 mm; TOSOH, Tokyo, Japan)
using 60% acetonitrile (isocratic) at a flow rate of

0.8 mLÆmin
)1
.
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