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Biochem. J. (2011) 437, 223–230 (Printed in Great Britain)
223
doi:10.1042/BJ20110292
Functional analysis of hyperthermophilic endocellulase from Pyrococcus
horikoshii by crystallographic snapshots
Han-Woo KIM and Kazuhiko ISHIKAWA1
National Institute of Advanced Industrial Science and Technology (AIST), Health Research Institute, 1-8-31, Midorigaoka, Ikeda, Osaka 563-8577, Japan, and Biomass Technology
Research Center, 3-11-32, Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046, Japan
A hyperthermophilic membrane-related β-1,4-endoglucanase
(family 5, cellulase) of the archaeon Pyrococcus horikoshii was
found to be capable of hydrolysing cellulose at high temperatures.
The hyperthermophilic cellulase has promise for applications in
biomass utilization. To clarify its detailed function, we determined
the crystal structures of mutants of the enzyme in complex with
either the substrate or product ligands. We were able to resolve
different kinds of complex structures at 1.65–2.01 Å (1 Å =
0.1 nm). The structural analysis of various mutant enzymes
yielded a sequence of crystallographic snapshots, which could
be used to explain the catalytic process of the enzyme. The
substrate position is fixed by the alignment of one cellobiose
unit between the two aromatic amino acid residues at subsites
+ 1 and + 2. During the enzyme reaction, the glucose structure
of cellulose substrates is distorted at subsite − 1, and the β1,4-glucoside bond between glucose moieties is twisted between
subsites − 1 and + 1. Subsite − 2 specifically recognizes the
glucose residue, but recognition by subsites + 1 and + 2 is loose
during the enzyme reaction. This type of recognition is important
for creation of the distorted boat form of the substrate at subsite
− 1. A rare enzyme–substrate complex was observed within the
low-activity mutant Y299F, which suggested the existence of a
trapped ligand structure before the formation by covalent bonding
of the proposed intermediate structure. Analysis of the enzyme–
substrate structure suggested that an incoming water molecule,
essential for hydrolysis during the retention process, might be
introduced to the cleavage position after the cellobiose product at
subsites + 1 and + 2 was released from the active site.
INTRODUCTION
has been provided by visualization of a covalent glycosyl-enzyme
intermediate by X-ray crystallography of enzyme–ligand complex
structures formed with saccharide-substrate analogues substituted
with fluoride at the 2-position of the glucopyranoside [8–10].
This covalent intermediate was also observed between a native
saccharide substrate and mutant enzyme [11–13]. Therefore the
double-displacement mechanism involving the formation of a
covalent intermediate has been generally accepted in this class
of enzymes. Structural data necessary to elucidate the catalytic
mechanism of hyperthermophilic endocellulase have long been
awaited. In a previous study, we prepared a protein crystal using
truncated forms of EGPh [14], and the protein structure of EGPh
was determined by X-ray crystallography at high resolution [15].
However, we were not able to obtain any useful X-ray diffraction
data of protein complexed with either substrate or product ligand.
In the present study, under different crystallization conditions,
we succeeded in preparing an alternative crystal for analysis
of the enzyme–ligand complex structure, in which substrate or
product ligand was competently bound to the active centre.
Three molecules of EGPh in the crystallographic asymmetric
unit were identified, allowing us to obtain many different types
of the complexed structure. Crystallographic snapshots of the
sequential events during the enzymatic reaction were obtained
from various mutant enzyme–ligand complex structures. These
snapshots described the detailed catalytic process of EGPh.
Using these structural data, we analysed the detailed role
of the subsite structure, as well as the catalytic mechanism of
hyperthermophilic endocellulase.
Cellulase is a key enzyme involved in the degradation of βglucan cellulose biomass. A hyperthermophilic cellulase would be
particularly useful in industrial applications. A hyperthermophilic
archaeon, Pyrococcus sp., was isolated from a hydrothermal
volcanic vent in the ocean [1,2]. Hyperthermophilic β-1,4endocellulases (endo-type cellulases) have been identified in
the genome sequences of several hyperthermophilic archaea.
The first hyperthermophilic endo-type cellulase (family 12)
was found in Pyrococcus furiosus; however, this enzyme
exhibited relatively low activity towards cellulose [3]. We
previously isolated a unique membrane-related hyperthermophilic
endocellulase from Pyrococcus horikoshii (termed EGPh; family
5) exhibiting relatively high activity towards cellulose [4,5].
A membrane-related hyperthermophilic β-1,4-glucosidase has
also been isolated from P. horikoshii [6]. Based on our current
knowledge, it appears that archaea use a primitive mechanism,
utilizing two enzymes to obtain glucose from cellulose.
The family 5 glycosyl hydrolase that belongs to clan GHA (glycoside hydrolase clan A) is a large superfamily with
divergent substrate specificity (http://www.cazy.org/GH5.html).
The superfamily has also been referred to as the 4/7 superfamily
because the three key residues are adjacent asparagine/glutamate
residues at the end of β-strand 4 and a glutamate residue at the end
of β-strand 7. Retention is the accepted catalytic mechanism of
the family 5 enzymes, although the fundamental mechanism has
been the subject of debate for many years. Evidence of the doubledisplacement mechanism originally suggested by Koshland [7]
Key words: cellulose, endocellulase, endoglucanase, hydrolytic
mechanism, hyperthermophile, Pyrococcus horikoshii.
Abbreviations used: EGAc, family 5 endocellulase E1 from Acidothermus cellulolyticus ; EGPh, hyperthermophilic endocellulase from Pyrococcus
horikoshii ; G2, cellobiose; G4, cellotetraose; PSA, phosphoric acid-swollen Avicel; rmsd, root mean square deviation; WT, wild-type.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2011 Biochemical Society
224
H.-W. Kim and K. Ishikawa
EXPERIMENTAL
RESULTS
Construction and preparation of truncated protein and site-directed
mutagenesis
Crystallization of the enzyme–ligand complex
The protein crystallization and structural analysis were conducted
using EGPhN5C5, which is the truncated protein lacking five
amino acid residues from both the N- and C-terminal ends
of EGPh (Gene ID PH1171, EC 3.2.1.4) [16]. Point-mutant
enzymes were prepared by site-directed mutagenesis using the
QuikChange® mutagenesis method (Stratagene). All mutant
genes were inserted into the expression vector pET11a (Novagen).
The constructed plasmids were introduced into Escherichia coli
strain BL21(DE3) for recombinant protein expression. Expression
and purification of the recombinant enzymes was carried out
following a method described previously [5,16].
Crystallization
The purified proteins were dialysed against 50 mM Tris/HCl
buffer (pH 8.0) and then concentrated to 20 mg·ml − 1 .
Crystallization was performed using the hanging-drop vapourdiffusion method. The drops consisted of equal volumes (1.5 μl)
of the protein and reservoir solutions. The crystal of the enzyme–
ligand complex was prepared from EGPhN5C5 using a reservoir
solution consisting of 1.5 M ammonium phosphate and 0.1 M
Mes buffer (pH 6.5). The crystals were obtained over a period of
approximately 3 days at 22 ◦ C. The crystal of the EGPhN5C5–
ligand complex was co-crystallized under the same conditions
using a final concentration of 100 mM cellobiose (G2). In the
case of the mutants (E201A, E342A and Y299F), the complex
with cellotetraose (G4) was obtained by soaking a crystal for 2 h
in the used reservoir solution containing 10 mM G4.
Data collection and processing
The crystals were collected with a CryoLoopTM (Hampton
Research) and immediately flash-cooled at 100 K in a nitrogen
cryostream. Diffraction data of crystals for the complex were
collected at BL38 and BL44, SPring-8 (Harima, Japan),
and processed and scaled using the HKL2000 and CCP4
(Collaborative Computational Project, Number 4, 1994) [17]
packages. All model-building stages were performed with
Coot [18]. The modelling of the structure of the enzyme–
ligand complex was performed with Coot, and the refinement
was performed with REFMAC5 [17] and CNS [19]. The
dictionary of ligand is derived from the HIC-Up database
(http://xray.bmc.uu.se/hicup/). After the incorporation of the
ligand model in the F o − F c omit map, refinement of the enzyme–
ligand complex was carried out using REFMAC5 or CNS. The
diffraction data statistics and the crystallographic refinement
statistics are summarized in Table 1. Figures were produced using
PyMOL (http://www.pymol.org).
In a previous study [14], we prepared the crystal of the truncated
protein EGPhC5 of EGPh lacking five amino acid residues from
the C-terminus, and determined the apo-enzyme structure [15].
However, we could not obtain X-ray diffraction data suitable for
determination of the structure of the enzyme–substrate ligand
complex. Failure to obtain suitable diffraction data might have
been caused by the presence of zinc ions tightly bound between
the two catalytic glutamate residues, which present an obstacle
for the entrance of ligand in the active site. In the crystal screening
using EGPhN5C5 (the truncated protein lacking five amino
acid residues from both the N- and C-terminal ends of EGPh)
[16], we could prepare a new crystal with reagent number 48
[2.0 M ammonium phosphate and 0.1 M Tris/HCl (pH 8.5)]
in the crystal screening kit (Hampton Research), thus optimizing
the crystallization conditions as described in the Experimental
section. The new crystal belongs to the space group C2 with
unit cell dimensions a = 162 Å (1 Å = 0.1 nm), b = 58 Å
and c = 138 Å, and β = 109 Å. We identified three molecules
of EGPhN5C5 in the crystallographic asymmetric unit. The
rmsd (root mean square deviation) of the Cα atom was less than
0.5 Å among the three molecules (hereafter referred as MolA,
MolB and MolC respectively), suggesting that they were almost
identical. These structures of EGPhN5C5 are referred to as
WT (wild-type) EGPh in the present paper. In the packing of the
crystal form, the speculative active site of MolA and MolB does
not exhibit sufficient space for binding of cello-oligosaccharide
substrate. In MolC, however, sufficient space for substrate binding
was observed between MolC and MolB.
We prepared crystals of the EGPhN5C5–cellobiose complex
(WT–G2) under these conditions, and determined its structure.
The structure was refined within 1.9 Å (Table 1). Unlike family
5 endocellulase E1 from Acidothermus cellulolyticus (hereafter
referred to as EGAc) [22], we did not observe an electron-density
map for any condensed cello-oligosaccharides in the active site
of WT–G2. This result was consistent with HPLC analysis of
the product from the reaction mixture containing G2 (results
not shown). In the structure of WT–G2, three protein molecules
(MolA, MolB and MolC) in the crystallographic asymmetric unit
exhibited a high F o − F c electron-density map corresponding to
G2 at subsites + 1 and + 2. Figure 1 shows the structure of
MolA, MolB and MolC. The supposed catalytic residues (Glu201
and Glu342 ) were located at the non-reducing end of the bound
G2. The G2 at subsites + 1 and + 2, [Glc( + 1) and Glc( + 2)],
was mainly stacked by Trp273 and Tyr304 . In the structure of G2
between Trp273 and Tyr304 , the non-polar surface was co-planar
with the hydrophobic part of Glc( + 1) and Glc( + 2). The electron
density corresponding to G2 at subsites − 2 and − 1 was poor
compared with subsites + 1 and + 2. However, the existence of
some substance at the subsites could be sufficiently recognized
by the F o − F c omit map contoured at 3.0 σ in MolA, MolB and
MolC (Figure 1).
Activity measurement
The hydrolytic activity of the enzymes toward 0.5% Avicel
(Merck) was determined by measuring the amount of reduced
sugars released from substrate at 85 ◦ C in 100 mM acetate buffer
(pH 5.5). The reduced sugars were determined by a modified
Somogi–Nelson method [20] using glucose as a standard.
PSA (phosphoric acid-swollen Avicel), used as a substrate,
was prepared by swelling in 85% phosphoric acid, and then
by removing phosphoric acid by filtration and centrifugation
according to the method described previously [21].
c The Authors Journal compilation c 2011 Biochemical Society
Michaelis complex structure of the catalytic residue mutants
To examine the enzyme–substrate complex (Michaelis complex)
structure, we prepared crystals of the inactive mutants, E201A
and E342A, in which Glu201 and Glu342 were replaced by alanine.
The crystals were soaked with G4 in the crystallization solution
as described in the Experimental section. The refined protein
structures revealed no distinct change in comparison with the
apo-enzyme structure.
Functional analysis of hyperthermophilic cellulase
Table 1
225
Statistics of data collection and refinement
Values for the highest-resolution shell are given in parentheses.
Statistic
Data collection
Wavelength (Å)
Space group
Unit-cell parameters (Å)
Matthews coefficient (Å 3 ·Da − 1 )
Solvent content (%)
Subunits per asymmetric unit
Resolution range (Å)
Number of observed reflections
Total number of unique reflections
<I/ σ (I )>
Multiplicity
R merge *
Completeness (%)
Refinement
Resolution used in refinement
R cryst (%)†
R free (%)‡
Rmsd of
Bond distance (Å)
Bond angle (◦ )
Average B factor of
Protein molecules (Å2 )
Ligand molecules (Å2 )
Ramachandran plot
In most favoured regions (%)
In disallowed regions (%)
PDB code
WT–G2
E201A–G4
E342A–G4
Y299F–G4
0.9
C2
a = 162.2, b = 58.4,
c = 138.3, β = 109.2
2.38
48.26
3
50–1.90 (1.97–1.90)
281105
96770
24.1 (3.1)
3.1 (2.5)
0.042 (0.210)
94.9 (85.9)
1.0
C2
a = 162.3, b = 58.3,
c = 138.3, β = 109.6
2.33
47.32
3
50–2.02 (2.10–2.02)
269527
83759
12.1 (2.7)
3.4 (3.1)
0.072 (0.287)
95.1 (82.1)
1.0
C2
a = 162.5, b = 58.6,
c = 139.1, β = 109.7
2.36
47.93
3
50–2.01 (2.05–2.01)
209856
82731
16.4 (2.6)
2.9 (2.2)
0.066 (0.328)
88.3 (77.8)
0.9
C2
a = 163.1, b = 58.4,
c = 138.2, β = 109.5
2.35
47.67
3
50–1.65 (1.68–1.65)
464216
147323
16.4 (3.5)
3.3 (2.6)
0.064 (0.308)
94.9 (84.8)
50.00–1.90 (2.02–1.90)
22.8 (35.4)
24.1 (36.2)
28.99–2.00 (2.13–2.00)
19.6 (25.0)
24.8 (33.5)
35.25–2.01 (2.14–2.01)
22.8 (37.6)
26.1 (40.7)
34.90–1.65 (1.69–1.65)
18.8(30.6)
22. 6 (33.7)
0.007
1.7
0.022
1.8
0.007
1.6
0.025
2.0
37.0
51.9
29.7
39.2
47.9
54.1
24.84
31.71
85.1
0.0
3AXX
85.1
0.0
3QHN
85.4
0.1
3QHM
98.0
0.0
3QHO
*R merge = hkl i |Ii (hkl ) − <I (hkl )>| hkl i Ii (hkl ), where Ii (hkl ) is the i -th intensity measurement of reflection hkl , including symmetry-related reflections, and <I (hkl )> is their average.
†R cryst = hkl |F o − F c |/ hkl F o , where F o and F c are the observed and calculated structure factor amplitudes of reflection hkl respectively.
‡R free is calculated as the R cryst , using F o that were excluded from the refinement (5% of the data).
In the structures of E201A–G4 and E342A–G4, four glucose
residues of G4 were clearly observed in the F o − F c omit map
from subsite − 2 to subsite + 2 in the active-site cleft of MolA
and MolB (Figure 2). In MolC, however, G4 was observed at
subsites − 4 to − 1, and G2 was exhibited at subsites + 1 to + 2
on the calculated electron-density map (Figure 2). The Glc( − 4)
of MolC was stacked by Arg44 of MolB in the crystal form that was
lying on Trp82 of putative subsite − 4 of MolC. In the mutational
analysis, the activity of the W82A mutant was 25% lower than
that of WT enzyme (Table 2).
In the other structures of E201A–G4 and E342A–G4, one G4
unit was observed along the active-site cleft, and each glucose
residue interacted with the stacking residues (Phe69 , Trp273 and
Tyr304 ) at subsites − 2, + 1 and + 2 respectively (Figure 2).
However, the conformations of Glc( − 1) at subsite − 1 were
significantly different in the structures of MolA. The substratebinding pattern in MolB of E201A–G4 was almost identical
with that in MolA and MolB of E342A–G4 (Figure 2). In these
structures, Glc( − 1) does not interact with the corresponding
stacking residue Trp377 . In the case of MolA of E201A–G4
(Figure 2), however, Glc( − 1) interacted with Trp377 at subsite
− 1, so that the sugar conformation was changed from the relaxed
chair form to the distorted boat (1,4 B) form of the β-anomer.
Figure 3 shows the hydrogen-bond interaction between Glc( − 1)
and the conserved residues (Glu201 , Tyr299 and Glu342 ) in the three
structures. In MolA and MolB of E342A–G4, the 2 -OH group of
Glc( − 1) hydrogen-bonds with the putative proton-donor oxygen
(O2) of Glu201 (Figures 2 and 3A). In MolB of E201A–G4, a
unique hydrogen bond between the 2 -OH group of the Glc( − 1)
and oxygen (O1) of Glu342 was observed (Figure 3B). In MolA
of E201A–G4, however, the 2 -OH group of Glc( − 1) hydrogenbonds with two oxygens (O1 and O2) of Glu342 (Figure 3C). The
sequence of (A)→(B)→(C) in Figure 3 suggests a sequence of
snapshots that represent the formation of the stable Michaelis
complex structure. During the enzyme reaction, the hydrogen
bond between the 2 -OH group of Glc( − 1) and the proton-donor
oxygen (O2) of Glu201 is shifted to another bond between the
2 -OH group of Glc( − 1) and Glu342 . In the following stage, a
stable complex structure (the distorted boat form) is formed by
hydrogen-bonding with Glu342 and Tyr299 (Figure 3C).
In the superimposed structures of three enzyme–ligand complex
structures (WT–G2, E342A–G4 and E201A–G4), the location
of Glc( + 1) and ( + 2) was slightly different, even though they
were inside the same subsite position (Figure 4). The position
of the bound G2 unit at Glc( + 1) and ( + 2) shifted towards
the active centre by the formation of the distorted boat form
of Glc( − 1) (Figure 4). Despite this positional shift of the G2
unit, a common hydrogen bond was observed between the 2 -OH
group of Glc( + 2) and the carboxy group of Gln306 (Figure 4). To
understand the role of Gln306 , we examined the activity of mutant
Q306A. The mutant Q306A exhibited only 25% of the enzymatic
activity exhibited by the WT against the substrate PSA (Table 2).
Michaelis complex structure of the mutant Y299F
In the structure of MolA in E201A–G4 (Figure 3C), Tyr299
is located within hydrogen-bond distance (2.97 Å) of the
c The Authors Journal compilation c 2011 Biochemical Society
226
H.-W. Kim and K. Ishikawa
Å. Furthermore, a rare hydrogen bond between the 2 -OH group
of Glc( − 1) and the carboxy group of Glu201 (2.80 Å) was found
in the structure of Y299F–G4 (Figure 6A).
Structural change in the subsite − 1 position by catalytic mutation
The overall G4 conformation of MolA in E201A–G4 was identical
with that of Y299F–G4, but Glc( − 1) of MolA in E201A–G4
showed a subtle shift (∼0.44 Å) towards Glu201 replaced by alanine
(Figure 5) that influences the glycosidic linkage oxygen of the
scissile bond in the substrate (G4) structure. The shift of Glc( − 1)
seems to result from the lack of a spatial barrier caused by the
absence of a side chain on the Glu201 residue.
In the structure of Y299F–G4, the 2 -OH group of Glc( − 1)
exhibited hydrogen-bonding with Glu201 and Glu342 , the key
catalytic residues. The 2 -OH group of Glc( − 1) showed
hydrogen-bonding with the carboxy group of Glu342 , and was
simultaneously located within hydrogen-bonding distance (2.84–
3.05 Å) of the proton-donor oxygen (O2) of Glu201 (Figure 6A).
As mentioned above, this rare hydrogen-bond pattern has not been
reported in any other glycosyl hydrolase.
DISCUSSION
Figure 1 Crystallographic snapshots of the EGPhN5C5–cellobiose (WT–
G2) complex
The active sites of three structures in the asymmetric unit are shown in divergent-eyed stereo
view. The F o − F c omit maps of the subsites − 2 to + 2 contoured at 3.0 σ were calculated
prior to incorporation of G2 at the subsites + 1 and + 2 in the model structures.
endocyclic oxygen (O5) of Glc( − 1), and simultaneously
contacts (2.55 Å) the nucleophilic oxygen (O1) of Glu342 .
To clarify a functional role for Tyr299 in the catalytic
action of EGPh, we determined the structure of Y299F
complexed with G4. The mutant Y299F exhibits slight
hydrolytic activity towards PSA, producing G2 (Table
2). The crystal structure of Y299F–G4 was resolved at
1.65 Å. In the active-site cleft of Y299F–G4, the whole G4
structure was clearly observed in the F o − F c omit map. Subsites
− 2 to + 2 were covered by the bound G4, and the structure of
Glc( − 1) also exhibited the distorted boat form similar to that
of MolA of E201A–G4 (Figures 5 and 6A). However, a strong
negative electron density was found in the scissile bond of the
substrate structure model from the final refinement (Figure 6B).
Figure 5 shows the superimposed structures from E201A–
G4(MolA), Y299F–G4 and WT–G2(MolA). The overall structure
of Y299F–G4 shows no significant difference with MolA of
E201A–G4 structure (Figure 5). In the superimposed structure of
Y299F–G4 and the other structures, however, a noticeable change
was observed in the χ angle of Glu201 (Figure 5). The difference
of the χ angle of Glu201 was also not observed in the structure
of Y299F. A noticeable change was induced by the binding of G4
in Y299F. The distance between the oxygen of the carboxy group
in Glu201 and the C1 atom of Glc( − 1) was estimated to be 3.10
c The Authors Journal compilation c 2011 Biochemical Society
In a previous study [15], we determined the crystal structure
of EGPhC5, but failed to obtain useful data to indicate the
structure of the enzyme–substrate or enzyme–product complex.
This seems to have been caused by the presence of zinc ions
tightly bound between the two catalytic glutamate residues,
which obstructed the entrance of ligand into the active-site cleft.
An alternative crystal of EGPhN5C5, which was prepared in
a crystallization solution without any bivalent metal cations,
was used for the analysis of the enzyme–ligand structures.
Unlike the previous crystal [14], this new crystal involved
three protein molecules in the crystallographic asymmetric
unit. These protein structures were almost identical, but their
active sites exhibited some differences, which allowed us to
obtain various crystallographic snapshots of the enzyme–ligand
complex structure. These snapshots aided our understanding of
the substrate-binding and catalytic mechanism of the enzyme.
Michaelis complex structure
In E201A–G4 and E342A–G4, the structures of MolC exhibited a
different substrate-binding pattern from those of MolA and MolB.
Two substrate molecules were separately bound on both sides of
the catalytic centre in MolC (Figure 2). This substrate-binding
pattern, which has not been observed in other family 5 enzymes,
revealed the additional subsites − 4 and − 3, which indicate that
the main substrate-binding site of EGPh stretches from subsite
− 4 to subsite + 2. That W82A exhibits low activity towards
PSA (Table 2) suggests that Trp82 functions as a stacking residue
at subsite − 4. It was also confirmed that Trp82 is conserved in
subfamily 1 of family 5 cellulases (results not shown). The finding
of the additional substrate-binding site (subsites − 3 and − 4)
was due to the sufficient space for substrate binding at the active
site of MolC in the crystal form. In both MolA and MolB, the
substrate-binding space (subsites − 3 and − 4) was blocked by
the interface of the neighbour molecule in the crystal form. The
structures of MolA and MolB in E342A–G4 (Figure 2) provide
an insight into the early stage of the substrate-binding process
(Figures 3 and 4). The result that Glc( − 1) in E342A–G4 exhibits
no stacking interaction with the corresponding aromatic residue
(Trp377 ) indicates that Glu342 is involved in the stacking interaction
between Trp377 and Glc( − 1). The structure of Glc( − 1) of MolB
Functional analysis of hyperthermophilic cellulase
Figure 2
227
Conformation of cellotetraose (G4) bound to the inactive mutants (E201A and E342A)
Left-hand panels in MolA show divergent-eyed stereo views of the model structures and F o − F c omit maps (contour level 3.0 σ ) calculated prior to incorporation of G4 in the model. MolC of
E342A–G4 also represents a F o − F c omit map (contour level 3.0 σ ) calculated without the contribution of G4 and G2. The structures of E201A–G4 and E342A–G4 are divided into two horizontal
sections respectively.
in E201A–G4 exhibits hydrogen-bonding between the 2 -OH
group of Glc( − 1) and the O1 atom of Glu342 . Interestingly, this
structure is similar to that of Glc( − 1) of MolA and MolB in
E342A–G4 (Figures 2 and 3). Therefore Glu342 appears to attract
a glucose moiety through the interaction between the 2 -OH group
of Glc( − 1) and Glu342 in the initial substrate-binding stage. After
this pulling action, the following stacking interaction of Glc( − 1)
and Trp377 seems to change Glc( − 1) from the normal chair form
into the distorted boat form (Figure 3). The resulting distorted
boat form would have a tendency to revert to the initial chair form
because of its unstable structural conformation.
The electron density at subsites + 1 and + 2 in the WT–
G2 structure was stronger than that of subsites − 1 and − 2,
indicating that subsites + 1 and + 2 have more stable and stronger
binding affinity towards the substrate than the other subsites.
Although this strong binding affinity exists at subsites + 1 and
c The Authors Journal compilation c 2011 Biochemical Society
228
Figure 3
H.-W. Kim and K. Ishikawa
Interactions of the Glc( − 1) and the catalytic residues in the active centre of the inactive mutants (E201A and E342A)
The refined structures from the enzyme–ligand complexes exhibit various hydrogen-bond interactions between the catalytically important residues (Glu201 , Tyr299 and Glu342 ) and the proximal glucose
unit Glc( − 1). The catalytic residues are shown as a cyan stick model. Possible hydrogen bonds are indicated by broken lines.
Figure 4
Comparison of the ligand-binding pattern in the active site
The structures of ligand from the three enzyme–ligand complexes [WT–G2 (green stick), E201A–G4 (yellow stick) and E342A–G4 (white stick)] were superimposed based on the conserved residues
of family 5 glycosyl hydrolases in the active centre. The superimposed structures are shown in divergent-eyed stereo view.
Table 2
Mutational analysis of the EGPhN5C5 enzyme
Enzyme
Relative activity (%) against PSA
EGPhN5C5 (WT)
W82A
Q306A
Y299F
W377A
100
75
25
<1
<1
+ 2, stretching of the glucose moiety at the position was observed,
as shown in Figure 4. In addition, since the position of Glc( − 2)
was not changed in these structures (Figure 4), the recognition of
glucose residue at subsite − 2 is specific. The movement of the
bound glucose residues at subsites + 1 and + 2, and the specific
binding at subsite − 2, are important for forming the distorted
boat form of Glc( − 1) at subsite − 1.
Characterization of the catalytic process
From the structural analysis, a narrower cleft was observed at
subsites + 1 and + 2, when compared with that of subsites − 1
and − 2 at the active site. At subsites + 1 and + 2, two aromatic
amino acid residues (Trp273 and Tyr304 ) stack one G2 unit and seem
to fix the position of the substrate. During the hydrolytic reaction
process, the glucose residue of cellulose is distorted at subsite
− 1, and the β-1,4-glucoside bond of cellulose is twisted between
c The Authors Journal compilation c 2011 Biochemical Society
Figure 5 Divergent-eyed stereo views of the superimposed structures
representing the slight conformational change from three enzyme–ligand
complexes
The structures of MolA of E201A–G4 (yellow stick), Y299F–G4 (white stick) and WT–G2 (green
stick) were superimposed based on the conserved residues. The glycosidic linkage oxygen of the
scissile bond in MolA of E201A–G4 is within hydrogen-bond distance with the proton-donor
residue, Glu201 , of WT–G2. In Y299F–G4, the χ angle of Glu201 was significantly changed
compared with that of WT–G2.
subsites − 1 and + 1 perpendicular to the cellulose fibre. The
final stage of the reaction is the release of the G2 from subsites
+ 1 and + 2.
Functional analysis of hyperthermophilic cellulase
Figure 6 Divergent-eyed stereo view of the active centre in the structure of
Y299F–G4
(A) Hydrogen-bond interaction between Glc( − 1) and the catalytic residues. The interaction
exhibits a rare hydrogen bond between the 2 -OH group of the Glc( − 1) and the proton-donor
oxygen (O2) of Glu201 . (B) Electron-density maps corresponding to Glc( − 1) and Glc( + 1) in
the structural analysis of Y299F–G4. Postive density of the F o − F c omit map, calculated prior
to incorporation of G4 in the model, is shown in green mesh (contour level 3.0 σ ). The negative
density of F o − F c map is in blue (contour level 3.0 σ ), which is generated from final refinement
of the model structure involving G4 in the active centre. The model refinement was performed
using REFMAC 5 TLS refinement.
That glucose and cellotriose were not released from the
reducing end of cello-oligosaccharides by EGPh indicates that
the affinity of subsite + 2 in EGPh is strong, and that the binding
of a glucose residue at subsite + 2 is important for catalytic
activity. The strong affinity of substrate (G2 unit) at subsites + 1
and + 2, and the strong negatively charged environment at the
catalytic centre (Glu342 ) characterize EGPh. This characteristic
may explain why EGPh has no condensation reaction activity,
unlike EGAc [22].
Role for Tyr299 in the retaining mechanism
The critical role of the conserved amino acid residues in family
5 enzymes were confirmed through mutational analysis [16].
Among these conserved residues, Trp377 and Tyr299 showed
structural and functional compatibility with family 5 cellulase
as well as family 18 chitinase [23]. The interaction between
Tyr299 and the endocyclic oxygen (O5) of Glc( − 1) observed
in the structure (Figure 3C) has been reported in the 2-fluoro
glucosyl-enzyme structure (covalent intermediate structure) of
family 1 myrosinase [24] and family 11 xylanase [25]. This
interaction was proposed to be an oxygen–oxygen interaction,
not a hydrogen bond, which might serve to stabilize the positively
229
charged oxocarbonium-like state through charge redistribution
with the negatively charged nucleophilic residue [25].
To clarify the functional role of Tyr299 , we constructed a Y299F
mutant and elucidated its structure complexed with G4. The
mutant Y299F exhibits slight hydrolytic activity towards G4.
However, the Y299F–G4 structure revealed a unique complex
structure involving the key catalytic residues (Glu201 and Glu342 )
and G4. The strong negative electron density that was generated
from the final refinement involving G4 indicates that the bound G4
was digested between Glc( − 1) and Glc( + 1). The structure in
Figure 6 represents the rare oxocarbonium-like structure trapped
by the carboxy group of Glu201 of the mutant Y299F. Interestingly,
the structure of Glc( − 1) with the distorted boat form showed a
rare hydrogen bond between the 2 -OH group and the protondonor oxygen (O2) of Glu201 , which causes a noticeable change
in the χ angle of Glu201 (Figures 5 and 6A). The χ angle of
Glu201 observed in the Y299F–G4 structure was not observed in
the Y299F structure. Furthermore, no change in the χ angle of
Glu201 was observed in the other mutants. This rare hydrogen
bond might be due to a change of the hydrogen-bond network
associated with protonation of Glu342 caused by the absence of the
OH group from Tyr299 . These results indicate that the structure of
Y299F–G4 is a unique intermediate structure in the hydrolysis
of G4 by Y299F. The pH activity profile of the Y299F mutant was
shifted towards alkaline pH, supporting the fact that protonation of
Tyr299 is important for catalytic action [16]. Thus the intermediate
states following the cleavage might not proceed in an ordered
sequence because of the hydrogen-bond interaction between the
2 -OH group of Glc( − 1) and the proton-donor oxygen (O2) of
Glu201 in Y299F. The results indicate that Tyr299 may be the protondonor residue and mediate the formation of the enzyme–substrate
intermediate during the catalytic process. Furthermore, it was
clarified that the OH group in Tyr299 is not necessarily important
for stabilizing the positively charged oxocarbonium-like state or
the distorted boat form. The carboxy group in Glu201 seems to
compensate for the role of Tyr299 .
Unlike the case of other enzymes [8–10,12,26], we could not
obtain any crystallographic snapshot of an intermediate structure
with a covalent bond between enzyme and ligand in the structural
analysis of EGPh. It seems to be difficult for EGPh to form an
intermediate because of the steric hindrance between the 2 -OH
group of Glc( − 1) and the carboxy group of Glu342 (Figure 3C).
Therefore, during the catalytic process, EGPh seems to attract
and position the substrate at the active site through the hydrogenbond interaction, as suggested by the sequence of (A)→(B)→(C)
(Figure 3). After formation of the enzyme–substrate complex (C),
positional change of Glu342 accompanied by the movement of
Tyr299 seems to be induced to form the proposed intermediate
with a covalent bond.
The structure of WT–G2 involves the final stage in the catalytic
events. In MolA, the electron density of G2 at subsites − 1 and
− 2 was recognized at 3.0 σ , although it was not clear (Figure 1).
This poor density is due to its disorder or heterogeneity. MolB and
MolC also have a planar and relatively strong unassigned electron
density at subsites − 1 and − 2. In MolB, we clearly observed one
water molecule in close proximity (2.50 Å) to Glu201 (Figure 1,
MolB). The position of the water molecule was compatible with
that of the O1 atom of the β-faced 1 -OH group of Glc( − 1)
in MolA (Figure 1, MolB and Figure 3C). This poor electron
density at subsites − 2 and − 1 in MolB seems to represent the
intermediate at the final stage. In these structures, the position of
the bound G2 at subsites + 1 and + 2 was clearly observed. The
position of the G2 shifts to the outside (approximately 2.10 Å),
compared with the structure of the E201A–G4, E342A–G4 and
Y299F–G4 complexes (Figure 5). Therefore, at the final stage, an
c The Authors Journal compilation c 2011 Biochemical Society
230
H.-W. Kim and K. Ishikawa
incoming water molecule essential for hydrolysis via the retaining
mechanism seems to be introduced to the cleavage site after the
bound product G2 moves towards the outside of the active cleft.
During the steady state of enzyme catalysis, the intermediate
form is easily changed to a more stable state. Therefore obtaining
protein crystals of the intermediate form is typically very difficult.
To address this problem, the intermediate structure has generally
been studied using chemical quenching methods involving the
use of enzyme mutations or substrate analogues [9,10,25,27].
However, the hyperthermophilic nature of EGPh allowed us to
study the intermediate forms more easily. The optimal temperature
for EGPh is >100 ◦ C. This is significantly higher than the
temperature required for crystallization (22 ◦ C). Thus, during
the co-crystallization of the enzyme and the ligands, the catalytic
reaction might appear to be suppressed by temperature quenching
[27], resulting in a rare intermediate form that was relatively well
maintained in crystalline form.
AUTHOR CONTRIBUTION
Han-woo Kim performed the experiments and designed the experiments. Kazuhiko Ishikawa
contributed to the experimental design and conceived the study.
ACKNOWLEDGEMENTS
We thank Ms Mai Nakatsuka for her help with the preparation of enzymes, and Dr Koich
Uegaki, Dr Tsutomu Nakamura, Dr Yasunobu Wada and Mr Yuji Kado for their kind
suggestions. The X-ray diffraction experiments were carried out with the approval of the
Japan Synchrotron Radiation Research Institute (Hyogo, Japan). We thank to Professor
Henry Nelson of Kinki University (Nara, Japan) for his helpful comments and critical
reading of this manuscript prior to submission.
FUNDING
This work was supported by the Japan Society for the Promotion of Science postdoctoral
fellowship programme.
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