Article
pubs.acs.org/JPCB
A Mechanistic Study of Trichoderma reesei Cel7B Catalyzed
Glycosidic Bond Cleavage
Yu Zhang, Shihai Yan,* and Lishan Yao*
Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, 266061,
China
S Supporting Information
*
ABSTRACT: An ONIOM study is performed to illustrate the
mechanism of Trichoderma reesei Cel7B catalyzed p-nitrophenyl lactoside hydrolysis. In both the glycosylation and
deglycosylation steps, the reaction proceeds in a concerted
way, meaning the nucleophilic attack and the glycosidic bond
cleavage occur simultaneously. The glycosylation step is rate
limiting with a barrier of 18.9 kcal/mol, comparable to the
experimental value derived from the kcat measured in this work.
The function of four residues R108, Y146, Y170, and D172,
which form a hydrogen-bond network involving the substrate,
is studied by conservative mutations. The mutants, including
R108K, Y146F, Y170F, and D172N, decrease the enzyme
activity by about 150−8000-fold. Molecular dynamics simulations show that the mutations disrupt the hydrogen-bond network,
cause the substrate to deviate from active binding and hinder either the proton transfer from E201 to O4(+1) or the nucleophilic
attack from E196 to C1(−1).
■
reesei Cel7A, the major exoglucanase, with ∼0.9 Å Cα rootmean-square deviation (rmsd) for matched residues (∼45%
sequence identity). One major difference between the two
structures is that the endoglucanase has a ∼50 Å cleft but the
exoglucanase has a tunnel with a similar length. The tunnel
facilitates processive cellulose degradations while the cleft
renders the enzyme ready to cleave cellulose randomly.
Shortening the loops of T. reesei Cel7A that cover the tunnel
increases the endo but decreases the exo activity,9 suggesting
that different active-site structures serve different purposes.
Though there has been no complex T. reesei Cel7B structure
available, several T. reesei Cel7A structures9−12 imply the cleft is
responsible for the cellulose chain binding and the glycosidic
bond cleavage. Different binding properties of Cel7A and cel7B
to cello-oligomer have been studied by molecular dynamics
simulations.13 It is also suggested that the binding of T. reesei
Cel7B to cellulose affects glucan clenching in the binding
cleft.14
Same as T. reesei Cel7A, T. reesei Cel7B is a retaining
glucosidase.15−17 It hydrolyzes glucan chains through a twostep mechanism, the glycosylation and deglycosylation steps.
Computational studies18−20 of the T. reesei Cel7A catalyzed
oligosaccharide hydrolysis showed that the glycosylation step
involves a proton transfer from the general acid (E217) to the
leaving group O4 and a nucleophile (E212) attack to the
INTRODUCTION
Cellulose, the most abundant renewable biomass source on
earth, is a polymer linked by unbranched β-1,4 glycosidic
bonds. Cellulose can be hydrolyzed to glucose which can then
be fermented to ethanol and other chemicals as substitutes for
products derived from fossil fuels. Cellulases are attractive
catalysts because they are more environmentally friendly than
the strong acid catalysts. However, cellulases have limitations
that the catalytic rate is slow, and high cellulase loads are
needed. Researches on cellulases are actively pursued to
understand the catalytic mechanism and enhance their
efficiency.
The filamentous fungus Trichoderma reesei (Hypocrea
jecorina) is of particular interest due to its remarkable ability
to secrete large amounts of cellulases that include exoglucanases
cleaving cellobiose from cellulose strands ends, endoglucanases
cleaving strands randomly, and β-glucosidases converting
soluble cellodextrins and cellobiose to glucose. These cellulases
usually consist of two domains, a large catalytic domain (CD)
and a small carbohydrate-binding module (CBM), combined
by an O-glycosylated linker peptide. The binding of the CBM
of T. reesei cellulases to cellulose enhances the activity by
increasing cellulases concentration on the cellulose surface.1−3
The CBM may also have a disruptive function against cellulose
as suggested by several studies.4−6 The glycosyl bond cleavage
is catalyzed by the CD domain. T. reesei Cel7B is one of the
major endoglucanases that accounts for 5−10% of the total
cellulases.7 The X-ray structure of apo T. reesei Cel7B CD has
been determined,8 which shows a great similarity to that of T.
© 2013 American Chemical Society
Received: April 22, 2013
Revised: June 8, 2013
Published: July 3, 2013
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The system was heated gently from 50 to 300 K in a 10 ps
NVT simulation, after which a 20 ps NPT (1 atm, 300 K) MD
simulation was performed. In both simulations, the heavy atoms
were restrained. Then, a unrestrained NPT MD simulation was
performed for 980 ps. Six MD snapshots, corresponding to the
time points of 200, 400, 600, 800, 980, and 1000 ps, were used
as the starting structures for NPT simulations with the length of
4 ns each. The R108K, Y146F, Y170F, and D172N mutant
simulations were run in the same way. In the protein CA and
substrate rmsd calculation, the last structure from 5000
unrestrained energy minimizations (following the restrained
minimization mentioned above) was selected as the reference.
ONIOM Calculation of the WT. The average structure of
the WT was calculated from six 4 ns trajectories. The snapshot
with the structure closest to the average was selected as the
initial model for QM/MM calculations, which were carried out
at ONIOM(B3PW91/6-31+G(d,p):AM1)44−47 level and performed with Gaussian 09 program.48 The model system
includes all the residues whose atoms are within 10 Å from
the O4(−1) of PNPL ligand, corresponding to a total of 36
residues (Figure 1). All MD water molecules were excluded.
anomeric carbon atom C1, whereas the deglycosylation involves
a proton transfer from a water molecule to E217 and a
hydroxide nucleophilic attack to C1. The importance of the two
catalytic residues is validated by the mutagenesis study that
E212Q and E217Q mutants decrease the T. reesei Cel7A
hydrolysis activity toward 2-chloro-4-nitrophenyl β-lactoside by
85- and 370-fold, respectively.11 A third acidic residue D214 is
thought to position E212 ready for the nucleophilic attack. The
sequence alignment shows that the corresponding general acid
and nucleophile in T. reesei Cel7B are E201 and E196, and the
assisting acidic residue is D198, all located in the cleft.8 Besides
these three residues, there are several other conserved ones in
the active site, especially in the −1 and −2 binding subsite. The
T. reesei Cel7A cello-oligosaccharide complex structures9−12
reveal that several but not all the residues interact with the
ligand directly. What are the roles of these residues? How do
they contribute to the catalysis? In this work, we adopt the
ONIOM21−23 and molecular dynamics simulation methods in
combination with the steady-state enzyme kinetics experiments
to study the mechanism of T. reesei Cel7B catalyzed pnitrophenyl lactoside (PNPL) hydrolysis. The ONIOM
method has been widely used to study enzymatic catalysis
mechanisms; e.g.,24−30 the MD simulation method has also
been successfully employed to study cellulases, including
Cel7A, Cel7B cellulose binding,14,31 Cel6A cello-oligomer
interaction,32 cellobiose release from Cel7A,33 etc.
Our study shows that similar to T. reesei Cel7A,18−20 a
concerted mechanism is adopted by Cel7B in both the
glycosylation and deglycosylation steps. Then, several activesite mutants in −1 and −2 binding subsites are studied. A large
variation of the catalytic activity decease is found for different
mutants from the steady-state kinetics study, highlighting the
importance of these residues. MD simulations of the mutants
show that though these residues do not involve in the catalysis
directly, they facilitate the reaction by restraining the substrate
for the proton transfer and nucleophilic attack.
■
METHODS AND MATERIALS
MD Simulation. The apo form T. reesei Cel7B crystal
structure (1EG1) reported by Jones8 was used as a starting
structure with the protonation states determined by a pKa
analysis.34−37 The ligand PNPL was added in the following way.
1EG1 was fitted to 8CEL, the T. reesei Cel7A cello-oligomer
complex crystal structure, by using PyMOL (Schrödinger,
LLC). The cello-oligomer coordinates were transferred to
1EG1 and then the ligand PNPL was inserted by fitting the
lactoside moiety to the glycosyl located at −2 and −1 subsites
and placing the p-nitrophenyl moiety at +1 subsite. The cellooligomer was then removed from the 1EG1 active site. The
simulations were carried out with the AMBER 11 molecular
dynamics package.38 The Amber force fields of FF99SB39 and
GAFF40 were employed to model the enzyme and the ligands.
The Antechamber module of Amber 11 was employed to
calculate BCC charges41,42 for the optimized PNPL structure
with AM1 Hamiltonian. Six Na+ ions were employed to
neutralize the system using the Amber LEAP tool. The complex
structure was immersed in an octahedral box of explicit
TIP3P43 water molecules, with a 12.0 Å distance between the
solvent box wall and the nearest solute atoms. Totally, there
were about 50 000 atoms in the system.
The details of MD simulations are as follows. Initially, the
system was minimized for 5000 steps with all heavy atoms
restrained by the harmonic potential (k = 10 kcal/(mol·Å2)).
Figure 1. ONIOM model of the WT Cel7B ES state. A total of 36
residues are included in the model. The residues in the high level,
including E201, E196, and PNPL, are depicted in balls and lines while
those in the low level are represented with lines.
The system was divided into two layers, as implemented in the
ONIOM method.49 The active-site amino acids, E201 and
E196 (modeled by propionic acid), as well as the moiety of
PNPL ligand located at −1 and +1 subsites were included in
the high layer and treated with the quantum mechanical
method. All remainder were included in the low layer treated
with AM1. The substrate complex was optimized with all the
atoms in the high layer as well as glycosyl at −2 subsite, D198
and N142 from the lower layer allowed relaxing. The optimized
substrate complex was used as the starting structure for the
subsequent study. For the glycosylation step, a 2D energy
surface was constructed by scanning C1(−1)···Oε2(E196) and
C1(−1)···O4(+1) distances independently from 1.46 to 3.16 Å.
For the deglycosylation step, C 1(−1)···O ε2 (E196) and
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Table 1. Primary Hydrogen Bond Distancesa (Å) in the Active Site of WT and Mutants
donor···acceptor
Nη2(R108)···O3(−2)
Nη2(R108)···O4(−2)
Oη(Y146)···O2(−2)
Oγ(S318)···O2(−2)
O3(−1)···Oδ1(D172)
O3(−1)···Oδ2(D172)
O2(−1)···Oε1(E196)
O6(−1)···Oδ1(D198)
Nη1(R108)···Oδ1(D35)
Oη(Y146)···Oη(Y170)
Oη(Y170)···Oδ2(D172)
Oδ2(D198)···Oε2(E196)
a
WT
3.07
3.19
2.80
2.90
3.28
3.38
2.68
3.09
2.86
2.78
3.66
2.58
±
±
±
±
±
±
±
±
±
±
±
±
0.30
0.29
0.14
0.21
0.70
0.68
0.16
0.79
0.19
0.12
1.07
0.09
R108K
4.78
5.12
2.83
2.86
3.07
3.08
2.61
5.49
3.68
2.82
2.83
2.58
±
±
±
±
±
±
±
±
±
±
±
±
Y146F
3.45 ±
3.70 ±
N/A
3.42 ±
3.73 ±
3.78 ±
3.32 ±
3.68 ±
3.11 ±
N/A
2.91 ±
2.62 ±
0.74
0.91
0.16
0.19
0.48
0.32
0.10
1.31
1.06
0.14
0.34
0.09
Y170F
0.84
0.67
0.71
1.66
1.58
1.59
1.25
0.48
0.34
0.28
4.63 ±
3.08 ±
4.27 ±
3.85 ±
4.49 ±
4.92 ±
3.01 ±
6.20 ±
3.46 ±
N/A
N/A
3.11 ±
0.54
0.35
0.44
0.80
0.91
0.75
0.35
0.90
0.75
0.91
D172N
3.04
3.15
2.82
2.89
4.66
3.52
3.77
3.17
2.95
2.83
4.44
2.60
±
±
±
±
±
±
±
±
±
±
±
±
0.27
0.25
0.14
0.19
0.73
0.62
0.77
0.82
0.29
0.15
0.49
0.10
The distance was averaged over six 4 ns MD trajectories.
substrate (final concentration 1, 1.8, 2.5, 3.5, 5, 6.5, 8 mM) in
100 mM acetate buffer (pH 4.0) with 0.3 μM WT Cel7B-CD.
Aliquots of 10 μL were taken at time intervals (0, 5, 8, 11, 14,
and 17 min) during the incubation and transferred immediately
to a microplate containing 190 μL of 1 M Na2CO3 to stop the
reaction. The amount of pNP liberated was determined by
measuring the absorbance at 405 nm using a microplate reader
(Ultrospec Visible plate reader II 96, GE Healthcare BioScience). The standard Michaelis−Menten equation was
applied to fit the kinetics kcat and Km values using an inhouse script. The amount of the added enzyme and the
sampling time were adjusted accordingly for the mutants to
reliably determine the reaction rates. All the experiments were
measured in triplicate.
C1(−1)···O(water) coordinates were selected and scanned
from 1.46 to 3.16 Å. The scanning distance interval was 0.10 Å.
To consider the effect of other residues in the vicinity of the
active site, the obtained stationary states from distance scanning
were reoptimized with more residues including R108, N142,
S144, Y146, S148, Y170, D172, Q174, and S318 unrestrained.
The solvation effect was included (at ONIOM(B3PW91/631+G(d,p):AM1) level) using the self-consistent reaction field
(SCRF) method employing the polarizable continuum model
(PCM)50,51 with a dielectric constant of 78.5 while the
structures of ES, TS1, EI(EI′), and EP were taken from
ONIOM(B3PW91/6-31+G(d,p):AM1) gas-phase optimizations. The effect of different density functionals was also
considered by optimizing the structures using B3LYP//631+G(d,p) for the high level while retaining AM1 for the low
level.
Cloning, Expression, and Purification. The DNA
encoding residues of the Cel7B catalytic domain (Cel7B-CD)
from T. reesei QM9414 and a 6*His tag at the C-terminus was
ligated with the vector pET-20b, which was digested with the
restriction enzymes, NdeI and HindIII. The ligation mixture
was transformed into an E. coli strain DH10B. The correct
coding sequence of the cloned catalytic domain of Cel7B gene
was verified by DNA sequencing. The expression vector (pET20b-Cel7B-CD) was then transformed into E. coli strain
origami (DE3). All the mutations were made by PCR-based
site-directed mutagenesis and verified by DNA sequencing. All
the mutants were expressed and purified in a similar way.
Briefly, 250 mL of LB medium containing 100 μg/mL
ampicillin was inoculated with a fresh colony of expression
strain origami (DE3) containing pET-20b-Cel7B-CD. The
culture was grown at 37 °C with vigorous shaking (∼200 rpm).
When the OD600 of the culture reached 0.8−1.2, a final
concentration of 1 mM of IPTG was added to induce the
expression of the protein at 16 °C and for 48 h. The cells were
harvested by centrifugation, washed twice with water,
resuspended in 100 mM NaAc buffer (pH4.0), and lysed by
ultrasound sonication. The lysed cells were centrifuged (9600g,
4 °C, 20 min) and the resulting supernatants were purified by
Ni-NTA affinity chromatography (Novagen). The purity was
determined by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE). Protein concentration was
measured by UV spectroscopy using an extinction coefficient
of 60 380 M−1 cm−1 at 280 nm.52
Kinetic Studies. Incubations of Lac-β-pNP as substrates
were carried out at 30 °C using a mixture (100 μL) containing
■
RESULTS AND DISCUSSION
MD of the WT Cel7B. The Cα rmsd of the protein obtained
from six 4 ns classical MD simulations, ranging around 1.6 Å,
illustrates the enzyme is fully equilibrated (Figure S1 in the
Supporting Information). The interactions between the ligand
PNPL and the protein are listed in Table 1. A total of eight
hydrogen bonds are formed between PNPL and the active site
residues, among which H-bonds OηH(Y146)···O2(−2, from
subsite −2), O2H(−1)···Oε1(E196), and OγH(S318)···O2(−2)
are relatively strong. Four weak hydrogen bonds were found,
with two between O3H(−2) and Oδ1,δ2(D172), and the other
two between Nη2H(R108)···O3,4(−2). Residues, including
R108, Y146, E196, and D172, are H-bonded with the substrate,
as well as other residues. For example, OηH(Y146) forms a Hbond with Oη(Y170) which is H-bonded as a donor to
Oδ(D172). All hydrogen bonds with PNPL are located in −2
and −1 subsites, suggesting the glycosyl group is well restrained
by the hydrogen-bond network (Figure 2) but the pnitrophenyl group is rather mobile. The dihedral angle C2−
C1−O5−C5 of the sugar ring at −1 subsite displays a two-state
behavior. The major state, with the occurrence frequency of
95.5%, has the average of −42.7 ± 8.9°, corresponding to the
4
C1 chair conformation. The minor state has the average of 58.7
± 11.1°, corresponding to the 1,4B boat conformation.
Catalytic Mechanism from ONIOM Calculations. For a
retaining GH enzyme such as T. Reesei Cel7B, the catalysis
involves two steps, glycosylation and deglycosylation. Two
distances C1(−1)···Oε2(E196) and C1(−1)···O4(+1) were
selected to describe the glycosylation step. The shortening of
C1(−1)···Oε2(E196) distance corresponds to the nucleophilic
attack from E196 to C1(−1) and the lengthening of
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C1(−1)···O(water) distances. The former describes the
glycosidic bond between ligand and protein whereas the latter
is an indicator for the nucleophilic attack from water. The
starting structure of the deglycosylation (defined as EI′)
resembles the EI state of the glycosylation step except that the
leaving group of p-nitrophenol is substituted by a water
molecule. Considering Km for PNPL is 14 mM, corresponding
to the binding energy of 2.5 kcal/mol for the substrate and the
main contribution is from the lactoside group due to the
hydrogen-bond interactions, the binding of p-nitrophenol
should be very weak. In other words, the energy difference
between EI′ and EI is very small, which is neglected in this
work. An early study by Bu et al.33 also showed that the
cellobiose release from Cel7B active site has a barrier of 5 kcal/
mol. So the release of p-nitrophenol, which is smaller and forms
fewer interactions with Cel7B, is facile. The water molecules
would fill in the space left by p-nitrophenol. After manually
adding one water molecule approaching the C1(−1) site, the
system was optimized. A hydrogen bond is formed between the
water molecule and E201 side-chain carboxyl group (EI′, Figure
4). Two energy minima are identified in Figure 5, with one
corresponding to EI′ and the other being the enzyme product
state (EP). EP, located on the lower right corner, has the
energy lower than that of the EI′ state by 13.9 kcal/mol. A
barrier of 10.5 kcal/mol is found along the MEP at the
C1(−1)···Oε2(E196) distance of 2.66 Å and the C1(−1)···O(water) distance of 2.16 Å. Figure 5 indicates that the transition
state geometrically resembles the product, predicting a late
transition state (TS2). Similar to the glycosylation step, a
concerted mechanism is adopted in the deglycosylation
reaction.
The energies for all the stationary states are listed in Table 2
and the structures are shown in Figure S2 in the Supporting
Information. In the structure optimizations, the atoms in the
high level and several atoms in the low level were allowed to
move while the rest of the system was fixed. To investigate
whether the relaxation of other residues in the vicinity of the
active site has a dramatic effect on the relative energies, we
reoptimized all the stationary states by allowing nine more
residues close to the active center to move (Methods and
Materials). The relative energies, listed in Table 2, show an
energy increase of 2−3 kcal/mol for TS1, EI, and TS2. Using
B3LYP for the high-level DFT has a minor effect on the relative
energies. Adding the solvation effect decreases the TS1 and EI
(EI′) energies by 2.3 and 6.7 kcal/mol, respectively. Thus,
allowing more residues to relax and including the solvation
term have some effects on the relative energies but do not
change the overall energy landscape dramatically. The overall
rate-limiting step is the glycosylation which has a barrier of 18.9
kcal/mol, comparable to the predicted free energy barrier (17.5
kcal/mol) of T. reesi Cel7A catalyzed cello-oligomer hydrolysis.18
Itinerary of Sugar Ring at the −1 Subsite. The sugar
ring at the −1 subsite of PNPL has the 4C1 chair conformation
in the ES state with the C2−C1−O5−C5 dihedral angle of
−54.8°. This sugar ring puckers along the glycosylation, via the
4
E geometry, into the 4H3 half-chair structure at the TS1
transition state with the C2−C1−O5−C5 dihedral of −6.7°.
When the glycosylation reaction is finished, the 4C1 chair
structure is recovered and the C2−C1−O5−C5 dihedral
decreases to −58.6° at the EI state. The C2−C1−O5−C5
dihedral changes slightly to −52.3° when a water molecule
substitutes the p-nitrophenol (EI′). In the step from EI′ to TS2,
Figure 2. H-bond network around the substrate PNPL. The H-bonds
are drawn with dashed lines.
C1(−1)···O4(+1) distance describes the glycosidic bond
cleavage. Figure 3 presents the contour plot of the potential
Figure 3. Contour plot of the potential energy surface for the
glycosylation step obtained at ONIOM (B3PW91/6-31+G(d,p):AM1)
level. The x-axis is the C1(−1)···Oε2(E196) distance, the reaction
coordinate for nucleophilic attack. The y-axis is the C1(−1)···O4(+1)
distance, describing the glycosidic bond cleavage or formation. The
reaction path from the reactant to the glycosylated intermediate is
shown with a green dashed line, with a barrier of 18.9 kcal/mol.
energy surface (PES) for this step. For the enzyme substrate
state (ES, Figure 4), C1(−1) and O4(+1) are covalently
bonded, while the Oε2(E196) is hydrogen bonded with the
C1H(−1) group. This state corresponds to the energy
minimum on the lower right corner of Figure 3. The other
energy-minimum state, on the top left corner, denotes the
glycosylated intermediate (EI, Figure 4) with a new covalent
C1(−1)−Oε2(E196) bond formed (the bond length of 1.465
Å). For EI, the C1(−1)−O4(+1) bond is cleaved, and the
proton is transferred from E201 to O4(+1) of PNPL.
Meanwhile, a strong hydrogen bond is formed between E201
and PNPL (Oε2(E201)···O4(+1) distance of 2.618 Å). The
relative energy of EI is almost equal to that of ES. The
minimum-energy path (MEP), shown with a dashed line in
Figure 3, connects two minima of ES and EI. The transition
state (TS1, Figure 4) locates between ES and EI states along
the MEP. For this transition state, the C1(−1)···Oε2(E196) and
C1(−1)···O4(+1) distances are 2.56 and 2.06 Å, respectively.
Thus, a concerted mechanism is adopted by the enzyme for the
glycosylation step.
The contour plot of the PES for the deglycosylation step is
shown in Figure 5, as a function of C1(−1)···Oε2(E196) and
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Figure 4. Schematic representation of different states along the Cel7B-catalyzed PNPL hydrolysis.
Table 2. Relative Energies of Stationary States Obtained at
Different Levels
B3PW91a
B3PW91(Relx)b
B3LYP(Relx)b
B3PW91(Solv)c
ES
TS1
EI (EI′)
TS2
EP
0.0
0.0
0.0
0.0
19.0
22.2
21.1
18.9
0.0
2.9
4.0
−3.8
10.5
12.8
12.1
11.7
−13.9
−14.9
−13.9
−14.7
a
The relative energies were obtained at ONIOM(B3PW91/631+G(d,p):AM1) level with the atoms in the DFT layer, −2 subsite
glycosyl group, residues D198 and N142 from the AM1 layer allowed
to move while the rest of the system was restrained. bONIOM(B3PW91/6-31+G(d,p):AM1) and ONIOM(B3LYP/6-31+G(d,p):AM1) were adopted for the energy calculations while allowing
more residues in the vicinity of the active site to relax (see main text).
c
PCM solvation model was incorporated to study the solvation effect.
Figure 5. Contour plot of the potential energy surface for the
deglycosylation step obtained at ONIOM (B3PW91/6-31+G(d,p):AM1) level. The x-axis is the C1(−1)···Oε2(E196) distance,
describing the glycosidic bond between the intermediate and the
protein. The y-axis is the C1(−1)···O(W) distance where O(W) is the
oxygen atom of a water molecule, describing the nucleophilic attack.
The reaction path from the glycosylated intermediate to the product is
shown using a green dashed line, with a barrier of 10.5 kcal/mol.
H-bonded to the ligand. A total of nine enzymes from glycoside
hydrolase family 7 (GH7) have their structures solved by the Xray crystallography (http://www.cazy.org/GH7_structure.
html), including Cel7A from Trichoderma harzianum,53 T.
reesei,11 Rasamsonia emersonii,54 and Heterobasidion annosum,55
Cel7B from Melanocarpus albomyces,56 T. reesei,8 Fusarium
oxysporum,57 and Humicola insolens,58 and Cel7D from
Phanerochaete chrysosporium.59 All structures share a similar
fold and active-site interactions. The sequence alignment of
these glycoside hydrolases (using ClustalW60) shows that the
active site has a conserved catalytic motif E-X-D-X-X-E, with
the first E as the nucleophile, the second E as the general acid,
and the D as the assisting acid. The alignment of the nine
sequences shows that various residues are strictly conserved in
the active site, including the catalytic acid/base E201, the
nucleophile E196, and several other residues in the −2, −1, and
+1 binding site in close contact with the ligand such as R108,
N142, Y146, Y170, D172, Q174, D198, H212, S318, and
the conformation of −1 subsite ring changes from 4C1 to 4H3
half-chair with the C2−C1−O5−C5 dihedral angle changing
from 60° to 3.5°. From TS2 to the product state, 4H3 changes
via E3 back to the 4C1 conformer with the C2−C1−O5−C5
dihedral of −59.7°.
Selection of Mutants. As revealed from the ONIOM
calculations, the general acid/base E201 and nucleophile E196
are essential for the catalysis. But the roles of other residues in
the active site are not well understood, including those directly
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binding of PNPL is weaker. The catalytic rate kcat of 525.7
min−1 corresponds to a barrier of 16.1 kcal/mol, as calculated
from the transition-state theory, which is comparable to that
from the ONIOM calculation. R108K decreases the kcat/Km by
about 130-fold and the main contribution is from kcat. In
comparison, Y146F and D172N reduce the activity by ∼1100fold and Y170F reduces it by ∼7700-fold. All the mutations are
rather conservative and only modify the residues slightly, but
cause a dramatic decrease of catalytic efficiency.
MD Simulation of the Mutants. To understand why
mutations cause such a drastic change of activity, a series of MD
simulations was performed for R108K, Y146F, Y170F, and
D127N in complex with the substrate PNPL. Overall, all-Cα
rmsd from the X-ray structure is similar for each enzyme
substrate complex (Figure S1 in the Supporting Information).
And the Cα root-mean-square fluctuation (rmsf) is also quite
similar (Figure S3 in the Supporting Information), suggesting
the mutations have a very minor effect on the overall structure
and dynamics. Further trajectory analyses suggest the active-site
interactions are modified by mutants (Figures S4 and S5 in the
Supporting Information).
R108K. Considering the side chain of R108 is about 10 Å
away from the C1(−1)···O4(+1) bond where the catalysis
occurs, it is surprising that R108K causes a dramatic change of
catalytic efficiency. In the simulation of WT Cel7B, R108 forms
two weak hydrogen bonds with O3(−2) and O4(−2) through
Nη2H, and a strong H-bond with Oδ1(D35) (Table 1). All three
hydrogen bonds disappear in the R108K mutant. The mutant
also disrupts the weak hydrogen bond between O6H(−1) and
Oδ1(D198) (Table 1). To gain more insight about the change,
two geometric parameters, r1, the C1(−1)···Oε2(E196) distance
and r2, the O4(+1)···Oε2(E201) distance, were monitored. The
former reflects the feasibility of the nucleophilic attack while the
latter is directly related to the proton transfer. For the WT
Cel7B, the distribution of (r1, r2) is shown in Figure 6, with an
W320. The interactions between the protein and the ligand are
mainly through the residue side chains. R108 and S318 are
hydrogen bonded to O3(−2); Y146 is H-bonded to O2(−2);
N142 is H-bonded to O6(−1); D172 is H-bonded to O3(−1);
W320 is in close contact with the −1 sugar ring through the van
der Waals interaction. Y170 has no direct contact with the
substrate but bridges Y146 and D172 through a hydrogen-bond
network. The sites selected for further characterization are
R108, Y146, Y170, and D172. The catalytic residues E196 and
E201, as well as the assisting residue D198, were not chosen
since they have been studied elsewhere in the T. reesei Cel7A
system.11 Conservative single mutations R108K, Y146F, Y170F,
and D172N, which were selected to minimize the structural
perturbation, were characterized experimentally and computationally. The results are discussed below.
Kinetic Rates. The initial reaction rate was measured at
different substrate concentrations and fitted to the Michaelis−
Menten equation (Table 3). The WT Cel7B has a kcat of 525.7
Table 3. Kinetic Constants for the Hydrolysis of PNPL by
the WT Cel7B and Mutants
WT
R108K
Y146F
Y170F
D172N
a
Km (mM)
kcat (min−1)
14.3 ±
17.1 ±
13.1 ±
N/A
21.1 ±
525.7 ± 48.3
4.77 ± 0.99
0.403 ± 0.08
N/A
0.71 ± 0.14
1.8
4.6
3.3
4.7
kcat/Km
(min−1mM−1)
rel
activitya
±
±
±
±
±
1
0.00766
0.00084
0.00013
0.00091
36.8
0.279
0.0308
0.0047
0.0337
1.3
0.017
0.0016
0.0004
0.0009
Relative activity is defined as the ratio (kcat/Km)mut/(kcat/Km)WT.
min−1, similar
(MeUmbL) and
which implies a
these substrates.
larger than that
to that of 4-methylumbelliferyl lactoside
cellotriose catalyzed by the same enzyme,52,61
similar catalytic mechanism is employed for
But the Km value of PNPL is about 10 times
for MeUmbL and cellotriose, suggesting the
Figure 6. Scattering plots for the distances of r1 and r2. r1 and r2 denote the distance of C1(−1)···Oε2(E196) and Oε2(E201)···O4 (+1), respectively.
Each plot includes 2400 dots extracted from the MD snapshots, corresponding to six 4 ns trajectories.
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in this mutant, consistent with the experimental findings that
Y170F has the lowest activity.
D172N. The carboxyl group of D172 is H-bonded with
O3(−1) and the hydroxyl of Y170 in the WT form. Mutating
D172 to an asparagine largely disrupts these two hydrogen
bonds. But the amine group of N172 forms a new hydrogen
bond with Oε1 of E196, the nucleophile, with the average heavy
atoms distance of 2.82 ± 0.25 Å. This H-bond pulls E196
slightly away from C1(−1), the nucleophilic attack site, revealed
by the average r2 distance of 3.41 Å which is ∼0.2 Å longer than
that of WT. It is expected that the hydrogen bond between
N172 and E196 pulls the electron density away from E196
carboxyl group so that Oε2(E196) becomes less negative
charged. These changes make the nucleophilic attack more
difficult for the mutant.
average of (3.22 Å, 3.47 Å). r1 is more symmetric and has a
much smaller standard deviation (0.16 Å) than that of r2 (0.79
Å). The r2 distribution has a positive skew. The skewed
distribution is corroborated with the distribution of the
substrate rmsd from the optimized docking model (Figure 7),
■
CONCLUSION
A catalytic mechanism is proposed for Cel7B-catalyzed PNPL
hydrolysis based on the ONIOM calculation. The reaction
proceeds through glycosylation and deglycosylation steps. In
the glycosylation step, along with the transfer of a proton from
E201 to O4(+1), the side-chain carboxyl of E196 attacks and
forms a covalent bond with C1(−1) and the glycosidic bond
C1(−1)−O4(+1) is cleaved. One product, p-nitrophenol, is
released. In the deglycosylation step, the hydroxide of a water
molecule attacks and bonds with C1(−1) and simultaneously
breaks the covalent bond between C1(−1) and Oε2(E196)
whereas the proton of the water transfers to E201. The other
product lactose is formed and the enzyme is regenerated for a
new catalytic cycle. The concerted mechanism is adopted in
both steps with the glycosylation step being rate-limiting.
Four conservative mutants including R108K, Y146F, Y170F,
and D172N display a low catalytic activity, in the order of
R108K > D172N > Y146F > Y170F. MD simulations show that
the mutants maintain the overall protein structure and
dynamics but alter the interactions in the active site. The
measured activity loss of the mutants is explained qualitatively
by larger C1(−1)···Oε2(E196) or O4(+1)···Oε2(E201) distances
in the simulation, suggesting the mutants either hinder the
proton transfer from E201 to O4(+1) or the nucleophilic attack
from E196 to C1(−1).
Figure 7. Histogram of the substrate PNPL rmsd from the reference
structure. Each histogram is built with a bin size of 0.2 Å, from 2400
MD snapshots (24 ns).
suggesting the skewness of r2 distribution is caused by the
substrate’s deviation from the starting structure. For the R108K
mutant, the average (r1, r2) values are (3.15 Å, 4.09 Å). The
average O4(+1)···Oε2(E201) distance is considerably larger
than the WT simulation, suggesting the proton transfer is more
difficult in the R108K mutant. Correspondingly, a larger
substrate RMSD is also observed for the mutant than the WT
(Figure 7).
Y146F. In the WT simulation, the hydroxyl group of Y146 is
H-bonded to O2(−2) and Y170. Removing this hydroxyl group
has a profound effect, not just limited to the two hydrogen
bonds. The MD trajectories of the mutant show most of the
hydrogen bonds in the active site are weakened or disrupted
(Table 1). The substrate average rmsd from the starting
structure is 1.83 ± 0.79 Å, considerably larger than that of WT,
suggesting in part of the MD trajectories the substrate drifts
away from the active site (Figure 7). This is reflected by the (r1,
r2) distribution as well (Figure 6). The average r1 and r2 values
are 3.58 and 3.81 Å, respectively. Both are larger than the WT,
suggesting the proton transfer and nucleophilic attack are more
difficult in the mutant.
Y170F. In the WT enzyme−substrate complex, Y170 does
not interact with the substrate directly. Similar to Y146F
mutant, most of the hydrogen bonds listed in Table 1 are
disrupted. The substrate displays a much larger rmsd from the
starting structure than WT (Figure 7) and the average r1 and r2
distances are 3.92 and 6.24 Å, respectively, the largest among all
the enzyme−substrate complexes. This observation suggests
the proton transfer and nucleophilic attack are the most difficult
■
ASSOCIATED CONTENT
S Supporting Information
*
The protein and ligand rmsds as well as the protein rmsfs of the
WT and mutants, Gaussian-optimized geometries of the WT at
various states, mutant structures of R108K, Y146F, Y170F, and
D172N, and the complete lists of refs 9, 31, 38, 48, and 60. This
material is available free of charge via the Internet at http://
pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected] (S.Y.); [email protected] (L.Y.).
Phone: 86 532 80662791 (S.Y.); 86 532 80662792 (L.Y.). Fax:
86 532 80662778 (S.Y.); 86 532 80662778 (L.Y.).
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
We are thankful to Supercomputing Center of Chinese
Academy of Sciences (CAS) for providing the computer
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Foundation for Outstanding Young Scientist in Shandong
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Scientific Research Foundation for the Returned Overseas
Chinese Scholars, State Education Ministry.
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