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.