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Mechanistic Study and Identification of Essential residues of Family 3  -Glucosidase.

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Presentation on theme: "Mechanistic Study and Identification of Essential residues of Family 3  -Glucosidase."— Presentation transcript:

1 Mechanistic Study and Identification of Essential residues of Family 3  -Glucosidase

2 Degradation of cellulose cellulose cellobiohydrolase cellodextrinscellobiose β-glucosidase glucose endo-β-glucanase Synergistic effect of cellulases

3 Assay method

4 Screening for  -glucosidase Acetobacter pasteurianus Neisseria subflava Micrococcus luteus Nocardia brevicatena Thermoactinomyces candidus Azospirillum brasilense Rhodococcus sp. Enterococcus faecalis Flavobacterium meningosepticum

5 Protein-Pak SP 40HR Mono-S Cation exchange chromatographic purification

6 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.

7 Characteristics of  -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 ℃.

8

9 Construction of genomic DNA library

10 Screening Method

11 10 kb Insert with  -Glucosidase Activity

12 Sub-cloning

13 Li, Y-K. * and Lee, J-A “Cloning and expression of β-glucosidase from Flavobacterium meningosepticum: a new member of family B β-glucosidase” Enzym. Microb. Technol. 24,

14

15 Substrate specificity

16 pH profile pK 1 =3.8~4.0, pK 2 =6.6

17 pK 1 pK 2

18 Anomeric configuration  -1,4-linked  -form  -form Inverting enzyme Retaining enzyme

19  -H  -H 35 min 25 min 15 min 5 min 0 min

20 Phenols H2OH2O

21 Active site affinity label +165 amu

22 Rate-limiting step? Phenols H2OH2O

23 Substrate Reactivity pKa

24 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 ” Catalytic mechanism of a family 3 b- glucosidase and mutagenesis study on its Asp-247” Biochem. J. 355,

25 ? TS 1 ? TS 2..

26 What does the transition state look like?

27 Secondary Kinetic Isotope Effect For S N 1-like : k D ~ For S N 2-like : k D ~ 1.0 k D (k H /k D )

28 ? TS 1 ? TS 2 k D =1.01, S N 2-like k D =1.19, S N 1-like..

29 Which are the essential residues of  -Glucosidase?

30 AF GMDVIHG 127WGRVSEGSGEDPY167VKHFALYGAPEG 241NGFIVTDY 454ANKADVVVLAIGETAELSGESSS AF LSDGPTG 114GGRLFEAYSEDPL148LKHLVANES-ET 222TGLVMSDW 567AAQADVAVVVVGLTEEEETESVD AL LSDGPNG 111NGRGFESFSEDST145IKHFVCNDM-ED 219KGTIISDW 559AKSVDCVILCVGLTAEWETEGED X VSDGPNG 111GGRGFESFSEDPY145VKHFVCNDL-ED 219DGMLMSDW 571AAKHDKAVLIIGLNGEWETEGYD M VTDGPNG 108NGRNFECYSEDPA142IKHFVANES-EI 216DGVVMSDW 542ARKSDIVLLLVGREGEWDTEGLD X MTDGPHG 117CGRNFEYFPEDPY151LKHFAANNQ-EH 226DGFVVSDW 401ASSSDVAVVFAGLPDEYESEGFD Z VSDGPHG 117SGRNFEYFSEDPY151LKHFAANNQ-EH 225EGIVVSDW 401ALKADVAVIFAGLPEHYECEGYD U VSDGPHG 125CGRNFEYFSEDPY159LKHFAANNQ-EH 233DGLVMSDW 404AMNADKVVVFAGLPDSFESEGFD D ETDAGQG 177NGRNFEYAGEDPL211LKHFVLNDQ-ET 285RGYVMSDW 475AAGADVALVFAN---QWIGEAND AB ISDAGLG 163GGRNFEYAGEDPL197LKHYAMNDL-ET 271PGFVMSDW 461ARAADVVVVYAT---QFTFEGMD AF ETDASLG 166NGRNFEYLGEDPL200VKHFSLNGQ-ET 274KGWVMSDW 466ARQSDIVILFAN---QWMSEGMD Y GTDGPAG 144AGRNFETFSEDPL178AKHYAANTQ-ET 251KGWVMSDW 544ARDSDVAVVFAY---DDGAETAD D AYDVVHG 167WGRASEGFGEDTY207VKHFAAYGAVEG 281KGITVSDH 499AKQADVVVAVVGESQGMAHEASS U AYDVLHG 191WGRASEGFGEDTY231VKHFAAYGAVEG 305KGITVSDH 523AKQSDVVVAVVGEAQGMAHEASS AF GMDVIHG 168WGRVSEGNGEDPF208VKHFALYGASEA 282DGFVVTDY 495AAGADVIVAALGESSEMSGESSS AF015915Flavobacterium meningosepticum (This study) D14068 Cellvibrio gilvus ATCC13127 AF005277Cellulomonas biazotea AB003689Acetobacter xylinus BPR2001 AL355920Schizosaccharomyces pombe AF090429Azospirillum irakense KBC1 X05918 Kluyveromyces fragilis Y14327 Saccharopolyspora erythraea M59852 Agrobacterium tumefaciens D86507 Salmonella typhimurium LT2 X15644Clostridium thermocellum ATCC U00007 Escherichia coli K12/BHB2600 Z94045 Clostridium stercorarium AF006658Bacteroides fragilis 638R (15 sequences are aligned.) U92808 Ruminococcus albus (15 sequences are aligned.)

31 Barley enzyme  -glucosidase Active Site Structural simulation Varghese JN, Hrmova M, Fincher GB, Structure 1999, 7,

32 Conserved Sequences D71 R129 K168 H169 D247 C3 C4 C2

33 Quick change Mutagenesis

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35 CD Spectra of wt and mutants WT  -glucosidase (X) D247N ( ○ )D247E ( ● )

36 LC/MS/MS spectrometry

37 Active site affinity label

38

39 Wild-type  -glucosidase labeled with 2F-DNPG following by pepsin digestion and HPLC column chromatography.

40 MS/MS daughter ion spectrum of the unlabeled peptide (m/z );---- m/z 1125 MS/MS daughter ion spectrum of the labeled peptide (m/z );---- m/z 1289

41

42 D247 in TDY sequence is the catalytic nucleophile!

43 Does E473 serve as general acid/base? Li, Y-K.*, Chir, J., Tanaka, S. and Chen, F-Y. (2002) Biochemistry, 41,

44

45 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

46 pH-profile of WT pH-profile of E473G

47 Intramolecular proton donor

48 Michaelis-Menten parameters of carboxyphenyl  -D-glucosides and derivatives catalyzed by Fbgl and E473G NaN 3 KmKm k cat K cat /K m Aryl-  -glucosides Enzyme(mM) (s -1 )(s -1 mM -1 ) WT E473G E473G WT E473G E473G WT E473G E473G200ND WT E473G E473G

49

50

51 Lys C-digested peptide (m/z ) Chir, J., Withers, S. G., Wan, C-F., and Li, Y-K.* (2002) Biochem. J. 365,

52 E473 is the acid/base catalyst of the enzyme!

53 Summary  A  -glucosidase containing 726 amino acid from F. meningosepticum was cloned and identified as a new member of GH-3 enzyme  GH-3  -glucosidase involves a two-step, double displacement mechanism.  D247 and E473 function as the nucleophile and the general acid/base catalyst, respectively.


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