Cellulose Chain Binding Free Energy Drives the Processive Move of Cellulases on
the Cellulose Surface
Yefei Wang †, Shujun Zhang †, Xiangfei Song, and Lishan Yao*,
Shandong Provincial Key Laboratory of Synthetic Biology, Laboratory of Biofuels, Qingdao
Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao,
266061, China
Supporting Information
Figure S1. Overlay of Tr. Cel7A (pdb:8CEL1, blue) and Tr. Cel7B (pdb: 1EG12, green). The
ligand cellononaose is shown with a stick model. The pair wise RMSD of two structures is 0.9 Å
for the matched C atoms.
Figure S2. The G1 and G2 released by Tr. Cel7B WT and the A335R mutant.
Figure S3. Correlation between the processivity (G2/G1 ratio) with Avicel as the substrate and the
predicted G. The best fitted line is G2/G1 = 1.09  0.19G, with a correlation coefficient R2 of
0.63 (excluding the outlier G225D).
Table S1. Specific activity of Tr. Cel7B WT and mutants on PNPL hydrolysis.
Enzyme
Specific
WT
(min •M )
122
A173S
17
S221K
15
A208Q
155
A222D
96
G225D
6
G230R
115
A335R
107
-1
activity
-1
Table S2.Catalytic activity of Tr. Cel7B WT and mutants against insoluble substrates Avicel and
FP as well as the soluble substrate PNPL.
FPa
Enzyme
G2
G1
（mM)
（mM)
Avicela
G2/G1
G2+G1
G2
G1
（mM)
（mM)
G2/G1
PNPLb
G2+G1
Specific
activity
(U/mg)
WT
0.44±0.01 0.38±0.01 1.16±0.01 0.82±0.01 0.34±0.01 0.35±0.01 0.97±0.01 0.69±0.01 1.95±0.10
A335R
0.50±0.02 0.32±0.03 1.56±0.04 0.82±0.04 0.39±0.02 0.28±0.01 1.39±0.02 0.67±0.02 1.70±0.08
G230R
0.45±0.02 0.26±0.01 1.73±0.02 0.71±0.02 0.36±0.01 0.23±0.03 1.57±0.03 0.59±0.03 1.84±0.11
G225D
0.24±0.01 0.10±0.01 2.40±0.01 0.34±0.01 0.22±0.01 0.11±0.01 2.00±0.01 0.33±0.01 0.09±0.00
A222D
0.46±0.01 0.30±0.02 1.53±0.02 0.76±0.02 0.40±0.01 0.37±0.01 1.08±0.01 0.77±0.01 1.54±0.09
A173S
0.34±0.01 0.18±0.01 1.89±0.01 0.52±0.01 0.32±0.01 0.22±0.01 1.45±0.01 0.54±0.01 0.27±0.01
A208Q
0.42±0.02 0.42±0.02 1.00±0.03 0.84±0.03 0.40±0.01 0.45±0.03 0.89±0.03 0.85±0.03 2.47±0.21
S221K
0.29±0.01 0.15±0.01 1.93±0.01 0.44±0.01 0.22±0.01 0.13±0.01 1.69±0.01 0.35±0.01 0.23±0.01
The reaction mixture contained 1.40 M Tr. Cel7B WT and mutants and the substrate of 10 g/L
Avicel or 10 g/L FP.
a.
b.
The reaction mixture contained 0.150.5 M Cel7B-CD and 1.67 mM PNPL.
Table S3. H-bonds analysis for the WT and mutants MD trajectories.
WT
A173S
S221K
A222D
G225D
G230R
A335R
(% )
(%)
(%)
(%)
(%)
(%)
(%)
-1@O6/N142@OD1
00
00
00
00
10
00
00
-1@O6/S144@OG
98  0
100  1
99  0
99  2
99  1
100  1
94  4
-1@O3/D172@OD1
91  1
77  1
49  4
68  8
32  3
77  2
72  2
-1@O3/D172@OD2
12  1
29  1
54  4
43  9
71  4
31  3
34  4
Q174@NE2/-1@O3
63  33
00
67  15
00
00
00
23  23
Q174@NE2/-1@O2
31  31
93  1
00
98  1
97  1
99  0
58  41
Q174@NE2/+1@O6
11
00
22
00
00
00
22
+1@O6/Q174@OE1
11  5
00
51
00
00
00
66
-1@O2/E196@OE1
100  0
99  0
11
100  0
100  1
100  0
100  0
-1@O2/E196@OE2
41
29  1
100  0
00
15  3
92
53
-1@O6/D198@OD1
00
00
00
00
00
00
44
E201@OE2/+1@O4
81  5
83  1
68  1
77  1
81  0
77  5
74  1
E201@OE2/-1@O5
56  9
56  2
72  1
62  2
66  3
63  3
67  0
-1@O6/E201@OE2
00
00
00
11
00
00
00
+1@O3/E201@OE1
98  1
96  0
99  0
96  1
98  1
99  1
97  2
T210@OG1/+1@O2
22
00
00
00
00
00
00
S221@OG/+2@O2
00
00
00
11
00
00
00
+2@O3/S221@O
00
00
00
10
00
00
00
K221@NZ/+2@O3
00
00
71
00
00
00
00
K221@NZ/+2@O2
00
00
11
00
00
00
00
K221@NZ/+1@O6
00
00
41
00
00
00
00
+2@O6/D222@OD2
00
00
00
29  2
00
00
00
+2@O6/D222@OD1
00
00
00
83
00
00
00
+1@O2/D222@OD2
00
00
00
25  3
00
00
00
G223@N/+1@O2
00
11
00
00
00
00
00
+2@O6/G223@O
11
11
00
00
00
00
00
+1@O2/G223@O
00
00
11
00
00
22
00
W320@NE1/-1@O6
97  2
96  1
97  1
95  2
96  1
97  1
93  4
R335@NH1/+2@O6
00
00
00
00
00
00
66  2
R335@NH2/+2@O6
00
00
00
00
00
00
14  7
+2@O2/Q325@OE1
21
66
00
11
41
44
21  17
+2@O6/W329@O
00
00
00
00
11
00
00
Total
746  1
762  4
722  18
800  9
757  4
755  5
827  45
Donor/Acceptor
a
b
a.
The donor corresponds to the hydrogen bond donor atom which has a proton attached and the
acceptor is the hydrogen bond acceptor atom. For example -1@O6/N142@OD1 means that the
O6 atom of the glycosyl unit at the -1 binding site forms a hydrogen bond with the OD1 of N142.
b.
The hydrogen bond is counted when the heavy atom distance is less than 3.5Å and the H-donoracceptor angle is smaller than 30. 800 snapshots from last 8 ns of 10ns MD simulation were used
for the analysis.
Table S4. The change of contacts between the enzyme and the glycosyl units at 1, +1, and +2
sites.
WT
A173S
S221K
A222D
G225D
G230R
A335R
N142
4.4  0.1
4.8  0.0
4.8  0.1
4.8  0.0
4.7  0.1
4.6  0.2
4.4  0.2
S144
5.0  0.0
5.0  0.0
5.0  0.0
5.0  0.0
5.0  0.0
5.0  0.0
5.0  0.0
Y146
5.7  0.0
5.8  0.0
5.2  0.1
5.9  0.0
5.7  0.1
5.8  0.2
5.5  0.1
Y170
4.4  0.0
4.9  0.0
5.2  0.1
4.8  0.1
5.6  0.1
5.0  0.1
4.9  0.0
D172
6.9  0.1
6.9  0.0
7.0  0.0
7.7  0.0
7.3  0.1
7.0  0.1
7.1  0.1
A173
3.3  0.1
3.7  0.1
3.2  0.0
3.2  0.1
3.0  0.0
3.0  0.1
3.1  0.1
Q174
16.9  1.5
20.0  0.8
13.3  0.3
21.3  0.3
20.4  0.1
21.7  0.3
16.0  0.4
E196
11.1  0.2
10.5  0.1
10.6  0.1
9.6  0.1
10.5  0.1
10.6  0.0
10.7  0.1
D198
10.2  0.4
10.1  0.0
9.7  0.0
8.5  0.1
10.3  0.1
10.2  0.1
9.5  0.2
E201
16.5  0.2
16.7  0.2
16.2  0.1
16.4  0.1
16.8  0.1
16.3  0.1
16.7  0.4
T210
7.6  0.1
7.3  0.3
7.6  0.0
7.4  0.0
7.6  0.0
7.6  0.0
6.5  0.9
H212
12.9  0.2
11.7  0.0
13.3  0.1
12.0  0.1
12.1  0.1
12.3  0.1
12.5  0.2
S221
7.7  0.8
7.4  0.6
16.5  0.3
9.2  0.6
7.0  0.0
8.7  0.6
5.6  2.9
A222
14.5  0.4
14.9  0.9
15.4  0.1
16.3  0.3
15.4  0.0
15.4  0.5
12.6  2.9
G223
4.5  0.1
4.2  0.2
3.9  0.2
4.5  0.2
4.3  0.1
4.0  0.1
3.6  0.5
G225
0.4  0.2
0.1  0.1
0.2  0.1
0.0  0.0
2.1  0.0
0.2  0.1
0.0  0.0
G230
0.0  0.0
0.0  0.0
0.0  0.0
0.0  0.0
0.1  0.0
0.4  0.3
0.0  0.0
S318
1.3  0.1
1.7  0.1
1.4  0.1
1.6  0.0
1.8  0.1
1.8  0.1
1.4  0.1
W320
8.9  0.1
9.9  0.3
8.2  0.0
9.8  0.1
10.1  0.1
9.8  0.2
10.5  1.0
Q325
0.8  0.2
1.4  1.3
0.2  0.0
0.8  0.1
0.9  0.1
0.9  0.5
4.6  3.5
W329
18.6  0.2
19.5  0.3
18.8  0.3
18.7  0.4
19.4  0.1
18.4  0.5
20.5  2.1
A335
0.0  0.0
0.0  0.0
0.0  0.0
0.0  0.0
0.0  0.0
0.0  0.0
4.9  0.8
161.2 
166.1 
165.3 
167.0 
169.6 
168.3 
165.0 
0.2
0.9
0.1
1.1
0.3
0.2
0.5
Total
References
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Mol. Biol. 1998, 275, 309-325.
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Srisodsuk, M.; Teeri, T. T.; Jones, T. A. The Crystal Structure of the Catalytic Core Domain of
Endoglucanase I from Trichoderma reesei at 3.6 Å Resolution, and a Comparison with Related
Enzymes. J. Mol. Biol. 1997, 272, 383-397.