Mechanistic Study and Identification of
Essential residues of Family 3
b-Glucosidase
Degradation of cellulose
cellulose
cellobiohydrolase
endo-β-glucanase
cellobiohydrolase
cellodextrins
cellobiose
β-glucosidase
glucose
Synergistic effect of cellulases
Assay method
HO
HO
HO
HO
O
HO
HO
O
OH
NO2 Enzyme
OH
+
O
NO2
HO
p-nitrophenyl-b-glucoside
Colorless
pK a =7.0
-O
NO2
Yellow, A 400 nm
OH
Screening for b-glucosidase
Acetobacter pasteurianus Neisseria
subflava
Micrococcus luteus
Nocardia brevicatena
Thermoactinomyces candidus Azospirillum
brasilense Rhodococcus sp.
Enterococcus faecalis
Flavobacterium meningosepticum
Cation exchange chromatographic purification
Mono-S
Protein-Pak SP 40HR
Lane 1: marker, Lane 2: crude cell
extract, Lane 3, 60 ~ 80% A.S. sat.,
Lane 4: SP column at pH 6.9, Lane 5:
Purified b-glucosidase from Mono-S at
pH 7.5.
Characteristics of b-glucosidase

No significant activity on hydrolysis of other
glycosides.

The estimated Mr. of the enzyme is 150 kDa by gel
filtration and 78 kDa by SDS-PAGE.

This dimeric enzyme has a pI= 9.0 and an optimal
activity at pH 5.0 and temperature of 50 ℃.
Construction of genomic DNA library
Genomic DNA
Plasmid DNA (pUC19)
BamH I: G GATCC
Sau3A I: GATC
Sucrose gradient
6-9 kb DNA fragment
CIP
dephosphorylation
Ligation
Transformation
Blue-white
selection
DNA library
Screening Method
HO
O
HO
OH
HO
CH3
OH
+
HO
O
HO
O
HO
O
CH3
OH
HO
O
Fluorescent product
ex :302 nm
10 kb Insert with b-Glucosidase Activity
Apa LI (178)
ALPHA
P(BLA)
Eco R I (397)
Apa LI (2367)
Ava I (413)
Xma I (413)
Sma I (415)
AP r
pUC19
2686 bp
Pst I (440)
Hin d III (448)
P(LAC)
ORI
Apa LI (1121)
BamHI
BamHI
10 kb DNA
Sub-cloning
Li, Y-K. * and Lee, J-A. 1999 “Cloning and expression of β-glucosidase from Flavobacterium
meningosepticum: a new member of family B β-glucosidase” Enzym. Microb. Technol. 24, 144-150.
Substrate specificity
HO
4
Y
2
HO
3
NO2
O
5
1
O
OH
Y
Glycosides
** Glucosides
Mannosides
Galactosides
Xylosides
Arabinosides
C2
e
a
e
e
e
C4
e
e
a
e
a
CH2OH
CH2OH
CH2OH
H
H
pH profile
pK1=3.8~4.0, pK2=6.6
EH2
pK 1
-
EH
pK 2
active spieces
2-
E
Anomeric configuration
b-1,4-linked
Inverting enzyme
a-form
Retaining enzyme
b-form
HO
O
HO
HO
OH
HO
HO
HO
O
OH
NO 2
O
H
phenol
a-H
25 min
15 min
5 min
0 min
H
Enzyme
HO
HO
HO
35 min
OH
b-H
O
OH
H
OH
H2O
Phenols
Active site affinity label
E473
E473
C
C
HO
O
O
O
H O2N
O
HO
O
HO
HO
F
O
O-
D247
HO
HO
-
O
O
Glucosylation
NO2
C
O
H
H
F
O
O
C
+165 amu
D247
S table Gl ucosyl-e n z ym e
in te rm e di ate
Rate-limiting step?
H2O
Phenols
Substrate Reactivity
HO
HO
O
O
HO
OH
X
pKa
4.1
5.2
5.4
6.5
7.2
7.2
8.2-10
X
2,4-dinitro
3,5-dinitro
2,5-dinitro
4-chloro-2-nitro
4-nitro
2-nitro
H, CN, Cl, CH3
The Bronsted plot & rate-limiting step
For good substrates (phenol pKa<7)
Deglucosylation
For Poor substrates (pKa>7)
Glucosylation
Li, Y-K. *, Chir, J. and Chen, F-Y. 2001 ” Catalytic mechanism of a family 3 bglucosidase and mutagenesis study on its Asp-247” Biochem. J. 355, 835-840.
c
c
o
O
H
HO
Gl u cosyl ati on
O
HO
O
HO
R
OH
HO
o
o
O
H
HO
HO
c o
D247
.
- O
O 
?
R
H
OH
o c oD247
TS1
TS= 1 (SN 2-l i ke )
o
HO
O
HO
c O
Gl u cosyl ati on
HO H
OH
HO
o c o
D247
Gl u cosyl -e n z ym e
in te rm e di ate
HO
O
H
c
De glu cosylation
-O c o
o
O
HO
OH
HO
HO
OH
o c oD247
.
b-gl u cose
?
H
O
HO
De glu cosylation
HO
OH
+ H
OH
o c o
D247
TS2
TS= 2 (SN 1-l i ke )
What does the transition
state look like?
HO
O
HO
HO
OH
O
HO
HO
HO
OH
E
+
O
HO
HO
OH
S N1-like
B
HO
+
S N 2-like
Secondary Kinetic Isotope Effect
HO
O2N
O
HO
HO
OH
For
HO
NO2
kD (kH/kD)
O
L
D
SN1-like: k ~
1.17
1.15-1.20
O2N
O
For SN2-like: kD ~ 1.0 1.19
HO
HO
OH
O
L
HO
HO
HO
O
OH
CN
O
L
1.01
c
c
o
O
H
HO
Gl u cosyl ati on
O
HO
O
HO
R
OH
HO
o
o
O
H
HO
HO
c o
D247
.
?
- O
O 
R
H
OH
o c o-
kD =1.01,
SN2-like
D247
TS1
TS= 1 (SN 2-l i ke )
o
HO
O
HO
c O
Gl u cosyl ati on
HO H
OH
HO
o c o
D247
Gl u cosyl -e n z ym e
in te rm e di ate
HO
O
H
c
De glu cosylation
o
O
HO
HO
?
HO
OH
OH
o c oD247
.
b-gl u cose
-O c o
H
O
HO
De glu cosylation
HO
OH
+ H
OH
o c o
D247
TS2
TS= 2 (SN 1-l i ke )
kD =1.19,
SN1-like
Which are the essential
residues of b-Glucosidase?
AF015915
69GMDVIHG 127WGRVSEGSGEDPY
167VKHFALYGAPEG 241NGFIVTDY 454ANKADVVVLAIGETAELSGESSS
AF005277
46LSDGPTG 114GGRLFEAYSEDPL
148LKHLVANES-ET 222TGLVMSDW 567AAQADVAVVVVGLTEEEETESVD
AL355920
43LSDGPNG 111NGRGFESFSEDST
145IKHFVCNDM-ED 219KGTIISDW 559AKSVDCVILCVGLTAEWETEGED
X05918
53VSDGPNG 111GGRGFESFSEDPY
145VKHFVCNDL-ED 219DGMLMSDW 571AAKHDKAVLIIGLNGEWETEGYD
M59852
39VTDGPNG 108NGRNFECYSEDPA
142IKHFVANES-EI 216DGVVMSDW 542ARKSDIVLLLVGREGEWDTEGLD
X15644
42MTDGPHG 117CGRNFEYFPEDPY
151LKHFAANNQ-EH 226DGFVVSDW 401ASSSDVAVVFAGLPDEYESEGFD
Z94045
42VSDGPHG 117SGRNFEYFSEDPY
151LKHFAANNQ-EH 225EGIVVSDW 401ALKADVAVIFAGLPEHYECEGYD
U92808
52VSDGPHG 125CGRNFEYFSEDPY
159LKHFAANNQ-EH 233DGLVMSDW 404AMNADKVVVFAGLPDSFESEGFD
D14068
97ETDAGQG 177NGRNFEYAGEDPL
211LKHFVLNDQ-ET 285RGYVMSDW 475AAGADVALVFAN---QWIGEAND
AB003689
94ISDAGLG 163GGRNFEYAGEDPL
197LKHYAMNDL-ET 271PGFVMSDW 461ARAADVVVVYAT---QFTFEGMD
AF090429
97ETDASLG 166NGRNFEYLGEDPL
200VKHFSLNGQ-ET 274KGWVMSDW 466ARQSDIVILFAN---QWMSEGMD
Y14327
81GTDGPAG 144AGRNFETFSEDPL
178AKHYAANTQ-ET 251KGWVMSDW 544ARDSDVAVVFAY---DDGAETAD
D86507
133AYDVVHG 167WGRASEGFGEDTY
207VKHFAAYGAVEG 281KGITVSDH 499AKQADVVVAVVGESQGMAHEASS
U00007
133AYDVLHG 191WGRASEGFGEDTY
231VKHFAAYGAVEG 305KGITVSDH 523AKQSDVVVAVVGEAQGMAHEASS
AF006658
110GMDVIHG 168WGRVSEGNGEDPF
208VKHFALYGASEA 282DGFVVTDY 495AAGADVIVAALGESSEMSGESSS
AF015915
Flavobacterium meningosepticum (This study)
D14068
Cellvibrio gilvus ATCC13127
AF005277
Cellulomonas biazotea
AB003689
Acetobacter xylinus BPR2001
AL355920
Schizosaccharomyces pombe
AF090429
Azospirillum irakense KBC1
X05918
Kluyveromyces fragilis
Y14327
Saccharopolyspora erythraea
M59852
Agrobacterium tumefaciens
D86507
Salmonella typhimurium LT2
X15644
Clostridium thermocellum ATCC 27405
U00007
Escherichia coli K12/BHB2600
Z94045
Clostridium stercorarium
AF006658
Bacteroides fragilis 638R
U92808
Ruminococcus albus
(15 sequences are aligned.)
Structural simulation
Barley enzyme
Varghese JN,
Hrmova M,
Fincher GB,
Structure 1999,
7,179-90.
Active
Site
b-glucosidase
Conserved Sequences
R129
D71
C4
C3
K168
C2
D247
H169
Quick change Mutagenesis
P CR to extend and
incorporate mutation
primers resulting in nick
circular strands
Denature plasmid and
annealing primers containing
desired mutation X
..
step 1
..
step2
xx
..
xx
step3
step4
xx
Mutant interested
o
xx
T ransform the resulting
annealed double-stranded
nicked DNA molecules
o
Digest paraental
DNA template
Michaelis Menten parameters for the hydrolysis of PNPG and
DNPG by wild-type Fbgl and mutants, pH 7.0, 37 ℃ .
r
r
Km
kcat
kcat
kcat/Km
kcat/Km
Substrate
Enzyme
(mM)
(s-1)
(%)
(M-1s-1) (%)
PNPG
DNPG
wild-type
0.49
31.9
100
65100
100
E132G
30
0.1
0.3
3
0.003
E136Q
1.08
23.4
73.4
21670
33.3
D137N
0.71
19.1
59.9
26770
41.0
H169A
0.48
0.02
0.06
41.7
0.06
H169S
0.33
0.1
0.31
303
0.47
D275N
0.79
20.1
62.8
25540
39.2
D458N
0.92
25.6
80
27830
43
E469Q
5.15
4.2
13.1
810
1.24
E473Q
0.49
0.009
0.03
17
0.03
E473G
0.02
0.006
0.02
340
0.52
D71N
0.22
0.43
1.19
1975
1.92
D71A
4.93
0.015
0.04
3.0
0.003
R129K
0.71
1.71
4.7
2408
2.35
R129A
3.47
1.91
5.24
519
0.51
K168G
0.62
0.12
0.34
199
0.19
E177Q
0.3
5.15
14.1
17170
17.0
D247N
1.5
0.0007
0.002
0.5
0.00045
E473Q
0.34
0.32
0.9
941
0.9
E437G
0.0004
0.012
0.03
30770
30.5
CD Spectra of wt and mutants
WT b-glucosidase (X) D247N (○) D247E (●)
LC/MS/MS
spectrometry
Active site affinity label
E473
E473
C
C
HO
O
O
O
H O2N
O
HO
O
HO
HO
F
O
O
D247
-
HO
HO
-
O
O
Glucosylation
NO2
C
O
H
H
F
O
O
C
D247
S table Gl ucosyl-e n z ym e
in te rm e di ate
Wild-type b-glucosidase labeled with 2F-DNPG following by pepsin
digestion and HPLC column chromatography.
MS/MS daughter ion
spectrum of the
unlabeled peptide (m/z
563 2+);---- m/z 1125
MS/MS daughter ion
spectrum of the labeled
peptide (m/z 645 2+);---m/z 1289
y-ions
unlabeled
912
labeled
(811)
(696)
533
432
(375)
262
148
976
(696)
533
432
375
261
148
1076
*
I V T D Y T G I N E
b-ions
unlabeled
213
314
429
592
693
750
863
978
labeled
213
314
593
756
857
914
1027
1142
y ion
CH CO
Rn
b ion
NH CH
Rn+1
D247 in TDY sequence is
the catalytic nucleophile!
Does E473 serve as
general acid/base?
Li, Y-K.*, Chir, J., Tanaka, S. and Chen, F-Y. (2002) Biochemistry, 41, 2751-2759.
E473
E473
C
C
HO
O
O
O
H
NO2
O
Glucosylation
O
HO
HO
OH
O
C
O
H
HO
OH
HO
O-
H
O
O
HO
NO2
-
O
O
C
D247
D247
Glu cosyl -e n z ym e
in te rm e di ate
HO
E473G
E473G
H
H
NO2
O
Glucosylation
O
HO
HO
OH
O
C
D247
NO2
O
-
HO
O
HO
HO
OH
O
O
C
D247
S table Gl ucosyl-e n z ym e
in te rm e di ate
Evidence of glucosyl-Enzyme intermediate
The apparent molecular mass of E473G mutant (1.8 mg/ml, 30 ml) after
incubation with 2’,4’-dinitrophenyl-b-D-glucopyranoside (20 mM)
for 5 min.
pH-profile of WT
pH-profile of E473G
Intramolecular proton donor
H
O
OH
HO
HO
O
O
H
O
OH
O
O
Michaelis-Menten parameters of carboxyphenyl b-D-glucosides and derivatives catalyzed by Fbgl and E473G
Aryl-b-glucosides
NaN3
Km
kcat
Kcat/Km
(mM)
(mM)
(s-1)
(s-1mM-1)
WT
0
30
0.012
0.0004
E473G
0
0.2
0.032
0.16
E473G
200
1.85
0.350
0.19
WT
0
0.12
0.004
0.33
E473G
0
0.13
0.0001
0.0008
E473G
200
1.89
0.0002
0.0001
WT
0
2.2
0.1
0.045
E473G
0
0.3
0.0003
0.001
E473G
200
ND
ND
ND
WT
0
0.72
1.17
1.63
E473G
0
0.1
0.003
0.03
E473G
200
4.5
0.014
0.003
Enzyme
HO
HO
HO
HOOC
O
HO
O
O
HO
HO
HO
O
H 3 C OC
HO
O
HO
HO
HO
O
HO
O
COOH
HO
HO
HO
O
O
HO
O
C OCH3
HO
E473G
E473G
H
H
NO2
O
Glucosylation
O
HO
HO
OH
O
C
D247
NO2
O
-
HO
HO
HO
N3-
O
OH
O
O
C
D247
S tabl e Gl u cosyl -e n z ym e
i n te rm e di ate
E473
E473
C
O
HO
-
O
O
NH
HO
HO
OH
CH2
C
O
C
Br
Alkylation
HO
HO
HO
O
OH
O
O
NH
CH2
C
O
S table in te rm e diate
Lys C-digested
peptide (m/z
1011 2+)
*
S G E S S S R A N I E I P Q A Q K
unlabeled b ions
labeled b ions
691
762
876
989
1119 1232 1329 1457 1528 1656
910 (981) 1096 1208 1338 1451 1548 1676 1747 1875
Chir, J., Withers, S. G., Wan, C-F., and Li, Y-K.* (2002) Biochem. J. 365, 857-863.
E473 is the acid/base catalyst
of the enzyme!
Summary
 A b-glucosidase containing 726 amino acid from F.
meningosepticum was cloned and identified as a
new member of GH-3 enzyme
 GH-3 b-glucosidase involves a two-step, double
displacement mechanism.
 D247 and E473 function as the nucleophile and
the general acid/base catalyst, respectively.