View
217
Download
0
Category
Preview:
Citation preview
Mechanistic Study and Identification of Essential
residues of Family 3 -Glucosidase
Degradation of cellulose
cellulose
cellobiohydrolase
cellobiohydrolase
cellodextrins cellobiose
β-glucosidase
glucose
endo-β-glucanase
Synergistic effect of cellulases
O
OOH
HO
NO2
HO
HO
p-nitrophenyl--glucoside
Enzyme
O
OHOH
HO
HO
HO
HO
NO2
+
O
NO2-
pK =7.0a
Colorless
Yellow, A 400 nm
Assay method
Screening for Screening for --glucosidaseglucosidase
Acetobacter pasteurianus Neisseria Acetobacter pasteurianus Neisseria subflava subflava
Micrococcus luteusMicrococcus luteus
Nocardia brevicatenaNocardia brevicatena
Thermoactinomyces candidus Azospirillum Thermoactinomyces candidus Azospirillum brasilense Rhodococcus sp.brasilense Rhodococcus sp.
Enterococcus faecalisEnterococcus faecalis
Flavobacterium meningosepticumFlavobacterium meningosepticum
Protein-Pak SP 40HR
Mono-S
Cation exchange chromatographic purification
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 -glucosidase from Mono-S at pH 7.5.
Characteristics of Characteristics of -glucosidase-glucosidase
No significant activity on hydrolysis of No significant activity on hydrolysis of other glycosides.other glycosides.
The estimated Mr. of the enzyme is 150 The estimated Mr. of the enzyme is 150 kDa by gel filtration and 78 kDa by SDS-kDa by gel filtration and 78 kDa by SDS-PAGE. PAGE.
This dimeric enzyme has a pI= 9.0 and an This dimeric enzyme has a pI= 9.0 and an optimal activity at pH 5.0 and temperature optimal activity at pH 5.0 and temperature of 50 ℃.of 50 ℃.
Construction of genomic DNA Construction of genomic DNA
librarylibrary
DNA library
Blue-white selection
Transformation
Ligation
CIP dephosphorylation6-9 kb DNA fragment
Sucrose gradient
BamH I: G GATCCSau3A I: GATC
Plasmid DNA (pUC19) Genomic DNA
Screening MethodScreening Method
Fluorescent product ex :302 nm
OO
OHHO
HO
HO
O
CH3
HO O
CH3
OOH
OHHO
HO
HO
+
pUC192686 bp
AP r
ALPHA
Bam HI Bam HI
P(BLA)
P(LAC)
ORI
Ava I (413)
Eco R I (397)
Hin d III (448)
Pst I (440)
Sma I (415)
Xma I (413)
Apa LI (178)
Apa LI (1121)
Apa LI (2367)
1 0 k b D N A
10 kb Insert with 10 kb Insert with -Glucosidase Activity-Glucosidase Activity
Sub-cloningSub-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.
Purification of Enzyme
Fermentation(LB medium
30Hrs, 37C,
170 rpm )
Sonication
Ammonium Sulfate(0~65% )
SP column
80604 02 00
0 .04
0 .02
0
Volumn ( ml )
A2
80
WT enzymeWT enzyme
Activity eluted at NaCl 0.1~0.150 M
93.2
66.455.642.736.5
26.6
All mutants were in high purity
Substrate specificitySubstrate specificity
54
3
2 1 O
HH
CH2OH
CH2OH
CH2OHGlycosides C2 C4 Glucosides e e Mannosides a eGalactosides e aXylosides e eArabinosides e a
NO2O
HO
HOY
OH
Y
**
pH profilepH profile
pK1=3.8~4.0, pK2=6.6
EH2 EH- E2-
active spieces
pK 1 pK 2
Anomeric configurationAnomeric configuration
-1,4-linked
-form-form
Inverting enzyme Retaining enzyme
-H-H35 min
25 min
15 min
5 min
0 min
O
OOH
HO
NO2
HO
HO
H
Enzyme
O
OHOH
HO
HO
HO
H
phenol
O
HOH
HO
HO
HO
OH
Phenols
H2O
Active site affinity labelActive site affinity label
-O H
H
C
OO
H
C
O O
E473E473
Stable Glucosyl-enzyme intermediate
O
HOHO
F
HO
Glucosylation
O2NOO
HOHO
F
HO
D247
NO2
C
O OC
O O
D247
- +165 amu
Rate-limiting step?Rate-limiting step?
Phenols
H2O
Substrate ReactivitySubstrate Reactivity
CH3
2,4-dinitro3,5-dinitro2,5-dinitro4-chloro-2-nitro4-nitro2-nitroH, CN, Cl,
O
X
X
O
HO
HO
OH
HO
pKa4.15.25.46.57.27.28.2-10
The Bronsted plot & rate-The Bronsted plot & rate-
limiting steplimiting step
For good substrates (phenol pKa<7) For good substrates (phenol pKa<7) DeglucosylationDeglucosylation
For Poor substrates (pKa>7) For Poor substrates (pKa>7) GlucosylationGlucosylation
Li, Y-K. *, Chir, J. and Chen, F-Y. 2001 ” Catalytic mechanism of a family 3 b-glucosidase and mutagenesis study on its Asp-247” Biochem. J. 355, 835-840.
HOHO
HOHO
HO
D247D247
c oo
D247
c oo
D247 D247
c oo
coo
o oc
-glucose
Deglucosylation O H
O H
HHO
O
HOHO H
Glucosyl-enzyme intermediate
Glucosylation
O HO
HOHO
OH
O
HOHO
OH
RR
OO
HOHO
OH
O H
O
HOHO
Glucosylation
TS 1=
TS 2=
Deglucosylation
O
(S 1-like)
N(S 2-like)
N
+
-
--
-
-
-
coO
H
coO
H
coO
H
co O -
H
coO
H
?
TS1
?
TS2
.
.
What does the What does the transition state look transition state look
like?like?
E
O
OHHO
HO
HO B
S 2-likeN
S 1-like
+
+
O
OHHO
HO
HO
O
OHHO
HO
HO
N
Secondary Kinetic Isotope EffectSecondary Kinetic Isotope Effect
For SN1-like: kD ~ 1.15-1.20
For SN2-like: kD ~ 1.0
kD (kH/kD)
1.17
1.19
1.01O
OOH
HOHO CN
HO
L
O
OOH
HOHO
O2NHO
L
O
OOH
HOHO
O2N
NO2
HO
L
HOHO
HOHO
HO
D247D247
c oo
D247
c oo
D247 D247
c oo
coo
o oc
-glucose
Deglucosylation O H
O H
HHO
O
HOHO H
Glucosyl-enzyme intermediate
Glucosylation
O HO
HOHO
OH
O
HOHO
OH
RR
OO
HOHO
OH
O H
O
HOHO
Glucosylation
TS 1=
TS 2=
Deglucosylation
O
(S 1-like)
N(S 2-like)
N
+
-
--
-
-
-
coO
H
coO
H
coO
H
co O -
H
coO
H
?
TS1
?
TS2
kD =1.01, SN2-like
kD =1.19, SN1-like
.
.
Which are the essential Which are the essential residues of residues of --Glucosidase?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.)(15 sequences are aligned.)
Barley enzyme
-glucosidase
Active Site
Structural simulationStructural simulation
Varghese JN, Hrmova M, Fincher GB, Structure 1999, 7,179-90.
Conserved Sequences
D71 R129
K168
H169D247
C3
C4C2
Quick change MutagenesisQuick change Mutagenesis
.. .. xx .. xx
xxo
oxx
step 1 step2
step3
step4
Denature plasmid and annealing primers containing desired mutation X
PCR to extend and incorporate mutation primers resulting in nick circular strands
Digest paraental DNA template
Transform the resulting annealed double-stranded nicked DNA moleculesMutant interested
Michaelis Menten parameters for the hydrolysis of PNPG and
DNPG by wild-type Fbgl and mutants, pH 7.0, 37 ℃.
Substrate Enzyme
Km (mM)
kcat
(s-1)
rkcat
(%)
kcat/Km (M-1s-1)
rkcat/Km
(%)
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
PNPG
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
DNPG
E437G 0.0004 0.012 0.03 30770 30.5
CD Spectra of wt and CD Spectra of wt and mutantsmutants
WT -glucosidase (X) D247N (○) D247E (●)
LC/MS/MS spectrometry
Active site affinity labelActive site affinity label
-O H
H
C
OO
H
C
O O
E473E473
Stable Glucosyl-enzyme intermediate
O
HOHO
F
HO
Glucosylation
O2NOO
HOHO
F
HO
D247
NO2
C
O OC
O O
D247
-
Wild-type -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
I V T D Y T G I N E
unlabeled
labeled
213 314 429 592 693 750 863 978
213 314 593 756 857 914 1027 1142
CH
Rn
CO NH CH
Rn+1
y ion
b ion
unlabeled
labeled
912 (811) (696) 533 432 (375) 262 148
1076 976 (696) 533 432 375 261 148
b-ions
y-ions
*
D247 in TDY sequence is the catalytic nucleophile!
Does E473 serve Does E473 serve as general as general acid/base?acid/base?
Li, Y-K.*, Chir, J., Tanaka, S. and Chen, F-Y. (2002) Biochemistry, 41, 2751-2759.
-O H
H
C
OO
H
C
O O
E473E473
Glucosyl-enzyme intermediate
O
HOHO
OH
HOGlucosylation
OO
HOHO
OH
HO
D247
NO2
NO2
C
O OC
O O
D247
-
E473GE473G
HH
Stable Glucosyl-enzyme intermediate
O
HOHO
OH
HOGlucosylation
OO
HOHO
OH
HO
D247
NO2
NO2
C
O OC
O O
D247
-
The apparent molecular mass of E473G mutant (1.8 g/l, 30 l) after incubation with 2’,4’-dinitrophenyl--D-glucopyranoside (20 mM) for 5 min.
Evidence of glucosyl-Enzyme intermediate
pH-profile of WT
pH-profile of E473G
Intramolecular proton Intramolecular proton
donordonor
OO
O O
OHOO
HOHO OH
H
H
Michaelis-Menten parameters of carboxyphenyl -D-glucosides and derivatives catalyzed by Fbgl and E473G
NaN3 Km kcat Kcat/Km
Aryl--glucosides Enzyme (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
OHO
HO
OHO
HOOC
HO
HO O
CO
HO
HO
OHO
H3CO
COOH
HO
OHO
HO
OHO
OHO
HO
OHO
HO
O
C
OHO
HO
OHO OCH3
E473GE473G
HH
Stable Glucosyl-enzyme intermediate
O
HOHO
OH
HOGlucosylation
OO
HOHO
OH
HO
D247
NO2
NO2
C
O OC
O O
D247
-
N3-
Stable intermediate
O
E473
CO
-
E473
Alkylation
O
NH
HOHO
OH
HO
C
CH2
O
Br
ONH
HOHO
OH
HO
C
CH2
O
C
O O
S G E S S S R A N I E I P Q A Q K
910 (981) 1096 1208 1338 1451 1548 1676 1747 1875
*
691 762 876 989 1119 1232 1329 1457 1528 1656unlabeled b ions
labeled b ions
Lys C-digested peptide (m/z 1011 2+)
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!
SummarySummary A A -glucosidase containing 726 amino acid -glucosidase containing 726 amino acid
from from F. meningosepticumF. meningosepticum was cloned and was cloned and identified as a new member of GH-3 enzymeidentified as a new member of GH-3 enzyme
GH-3GH-3-glucosidase involves a two-step, -glucosidase involves a two-step, double displacement mechanism.double displacement mechanism.
D247 and E473 function as the nucleophile D247 and E473 function as the nucleophile and the general acid/base catalyst, and the general acid/base catalyst, respectively. respectively.
Recommended