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Mechanistic Study and Identification of Essential residues of Family 3 b -Glucosidase

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.

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Mechanistic Study and Identification of Essential residues of Family 3 b -Glucosidase

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

  2. Degradation of cellulose cellulose cellobiohydrolase endo-β-glucanase cellobiohydrolase cellodextrins cellobiose β-glucosidase glucose Synergistic effect of cellulases

  3. Assay method

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

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

  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 b-glucosidase from Mono-S at pH 7.5.

  7. 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 ℃.

  8. Construction of genomic DNA library

  9. Screening Method

  10. 10 kb Insert with b-Glucosidase Activity

  11. Sub-cloning

  12. 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.

  13. Substrate specificity

  14. pH profile pK1=3.8~4.0, pK2=6.6

  15. pK 1 pK 2

  16. Anomeric configuration b-1,4-linked Inverting enzyme Retaining enzyme a-form b-form

  17. a-H b-H 35 min 25 min 15 min 5 min 0 min

  18. H2O Phenols

  19. Active site affinity label +165 amu

  20. Rate-limiting step? H2O Phenols

  21. pKa 4.1 5.2 5.4 6.5 7.2 7.2 8.2-10 Substrate Reactivity

  22. 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 b-glucosidase and mutagenesis study on its Asp-247”Biochem. J. 355, 835-840.

  23. ? . TS1 ? . TS2

  24. What does the transition state look like?

  25. Secondary Kinetic Isotope Effect kD (kH/kD) 1.17 1.19 1.01 For SN1-like:kD~ 1.15-1.20 For SN2-like:kD~ 1.0

  26. ? kD=1.01, SN2-like . TS1 ? kD=1.19, SN1-like . TS2

  27. Which are the essential residues of b-Glucosidase?

  28. AF015915 69GMDVIHG 127WGRVSEGSGEDPY 167VKHFALYGAPEG 241NGFIVTDY 454ANKADVVVLAIGETAELSGESSS AF00527746LSDGPTG 114GGRLFEAYSEDPL 148LKHLVANES-ET 222TGLVMSDW 567AAQADVAVVVVGLTEEEETESVD AL35592043LSDGPNG 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.)

  29. Structural simulation Barley enzyme • Varghese JN, Hrmova M, Fincher GB, Structure 1999, 7,179-90. Active Site b-glucosidase

  30. Conserved Sequences R129 D71 C4 C2 C3 K168 D247 H169

  31. Quick change Mutagenesis

  32. CD Spectra of wt and mutants WT b-glucosidase (X) D247N (○) D247E (●)

  33. LC/MS/MS spectrometry

  34. Active site affinity label

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

  36. 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

  37. D247 in TDY sequence is the catalytic nucleophile!

  38. Does E473 serve as general acid/base? Li, Y-K.*, Chir, J., Tanaka, S. and Chen, F-Y. (2002) Biochemistry, 41, 2751-2759.

  39. 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.

  40. pH-profile of WT pH-profile of E473G

  41. Intramolecular proton donor

  42. Michaelis-Menten parameters of carboxyphenyl b-D-glucosides and derivatives catalyzed by Fbgl and E473G NaN3 Km kcat Kcat/Km  Aryl-b-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

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