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. 2001 May 1;355(Pt 3):835–840. doi: 10.1042/bj3550835

Catalytic mechanism of a family 3 beta-glucosidase and mutagenesis study on residue Asp-247.

Y K Li 1, J Chir 1, F Y Chen 1
PMCID: PMC1221801  PMID: 11311148

Abstract

A family 3 beta-glucosidase (EC 3.2.1.21) from Flavobacterium meningosepticum has been cloned and overexpressed. The mechanistic action of the enzyme was probed by NMR spectroscopy and kinetic investigations, including substrate reactivity, secondary kinetic isotope effects and inhibition studies. The stereochemistry of enzymic hydrolysis was identified as occurring with the retention of an anomeric configuration, indicating a double-displacement reaction. Based on the k(cat) values with a series of aryl glucosides, a Bronsted plot with a concave-downward shape was constructed. This biphasic behaviour is consistent with a two-step mechanism involving the formation and breakdown of a glucosyl-enzyme intermediate. The large Bronsted constant (beta=-0.85) for the leaving-group-dependent portion (pK(a) of leaving phenols >7) indicates substantial bond cleavage at the transition state. Secondary deuterium kinetic isotope effects with 2,4-dinitrophenyl beta-D-glucopyanoside, o-nitrophenyl beta-D-glucopyanoside and p-cyanophenyl beta-D-glucopyanoside as substrates were 1.17+/-0.02, 1.19+/-0.02 and 1.04+/-0.02 respectively. These results support an S(N)1-like mechanism for the deglucosylation step and an S(N)2-like mechanism for the glucosylation step. Site-directed mutagenesis was also performed to study essential amino acid residues. The activities (k(cat)/K(m)) of the D247G and D247N mutants were 30000- and 200000-fold lower respectively than that of the wild-type enzyme, whereas the D247E mutant retained 20% of wild-type activity. These results indicate that Asp-247 is an essential amino acid. It is likely that this residue functions as a nucleophile in the reaction. This conclusion is supported by the kinetics of the irreversible inactivation of the wild-type enzyme by conduritol-B-epoxide, compared with the much slower inhibition of the D247E mutant and the lack of irreversible inhibition of the D247G mutant.

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Selected References

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  1. Barras F., Bortoli-German I., Bauzan M., Rouvier J., Gey C., Heyraud A., Henrissat B. Stereochemistry of the hydrolysis reaction catalyzed by endoglucanase Z from Erwinia chrysanthemi. FEBS Lett. 1992 Mar 30;300(2):145–148. doi: 10.1016/0014-5793(92)80183-h. [DOI] [PubMed] [Google Scholar]
  2. Bauer M. W., Kelly R. M. The family 1 beta-glucosidases from Pyrococcus furiosus and Agrobacterium faecalis share a common catalytic mechanism. Biochemistry. 1998 Dec 8;37(49):17170–17178. doi: 10.1021/bi9814944. [DOI] [PubMed] [Google Scholar]
  3. Bause E., Legler G. Isolation and amino acid sequence of a hexadecapeptide from the active site of beta-glucosidase A3 from Aspergillus wentii. Hoppe Seylers Z Physiol Chem. 1974 Apr;355(4):438–442. doi: 10.1515/bchm2.1974.355.1.438. [DOI] [PubMed] [Google Scholar]
  4. Béguin P., Aubert J. P. The biological degradation of cellulose. FEMS Microbiol Rev. 1994 Jan;13(1):25–58. doi: 10.1111/j.1574-6976.1994.tb00033.x. [DOI] [PubMed] [Google Scholar]
  5. Castle L. A., Smith K. D., Morris R. O. Cloning and sequencing of an Agrobacterium tumefaciens beta-glucosidase gene involved in modifying a vir-inducing plant signal molecule. J Bacteriol. 1992 Mar;174(5):1478–1486. doi: 10.1128/jb.174.5.1478-1486.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dale M. P., Ensley H. E., Kern K., Sastry K. A., Byers L. D. Reversible inhibitors of beta-glucosidase. Biochemistry. 1985 Jul 2;24(14):3530–3539. doi: 10.1021/bi00335a022. [DOI] [PubMed] [Google Scholar]
  7. Dale M. P., Kopfler W. P., Chait I., Byers L. D. Beta-glucosidase: substrate, solvent, and viscosity variation as probes of the rate-limiting steps. Biochemistry. 1986 May 6;25(9):2522–2529. doi: 10.1021/bi00357a036. [DOI] [PubMed] [Google Scholar]
  8. Dan S., Marton I., Dekel M., Bravdo B. A., He S., Withers S. G., Shoseyov O. Cloning, expression, characterization, and nucleophile identification of family 3, Aspergillus niger beta-glucosidase. J Biol Chem. 2000 Feb 18;275(7):4973–4980. doi: 10.1074/jbc.275.7.4973. [DOI] [PubMed] [Google Scholar]
  9. Day A. G., Withers S. G. The purification and characterization of a beta-glucosidase from Alcaligenes faecalis. Biochem Cell Biol. 1986 Sep;64(9):914–922. doi: 10.1139/o86-122. [DOI] [PubMed] [Google Scholar]
  10. Febbraio F., Barone R., D'Auria S., Rossi M., Nucci R., Piccialli G., De Napoli L., Orrù S., Pucci P. Identification of the active site nucleophile in the thermostable beta-glycosidase from the archaeon Sulfolobus solfataricus expressed in Escherichia coli. Biochemistry. 1997 Mar 18;36(11):3068–3075. doi: 10.1021/bi962496w. [DOI] [PubMed] [Google Scholar]
  11. Grover A. K., Cushley R. J. Studies on almond emulsin beta-D-glucosidase. II. Kinetic evidence for independent glucosidase and galactosidase sites. Biochim Biophys Acta. 1977 May 12;482(1):109–124. doi: 10.1016/0005-2744(77)90359-x. [DOI] [PubMed] [Google Scholar]
  12. Gräbnitz F., Rücknagel K. P., Seiss M., Staudenbauer W. L. Nucleotide sequence of the Clostridium thermocellum bgIB gene encoding thermostable beta-glucosidase B: homology to fungal beta-glucosidases. Mol Gen Genet. 1989 May;217(1):70–76. doi: 10.1007/BF00330944. [DOI] [PubMed] [Google Scholar]
  13. Henrissat B. A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J. 1991 Dec 1;280(Pt 2):309–316. doi: 10.1042/bj2800309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Henrissat B., Bairoch A. New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J. 1993 Aug 1;293(Pt 3):781–788. doi: 10.1042/bj2930781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hrmova M., Harvey A. J., Wang J., Shirley N. J., Jones G. P., Stone B. A., Høj P. B., Fincher G. B. Barley beta-D-glucan exohydrolases with beta-D-glucosidase activity. Purification, characterization, and determination of primary structure from a cDNA clone. J Biol Chem. 1996 Mar 1;271(9):5277–5286. doi: 10.1074/jbc.271.9.5277. [DOI] [PubMed] [Google Scholar]
  16. Hrmova M., MacGregor E. A., Biely P., Stewart R. J., Fincher G. B. Substrate binding and catalytic mechanism of a barley beta-D-Glucosidase/(1,4)-beta-D-glucan exohydrolase. J Biol Chem. 1998 May 1;273(18):11134–11143. doi: 10.1074/jbc.273.18.11134. [DOI] [PubMed] [Google Scholar]
  17. Hughes M. A., Brown K., Pancoro A., Murray B. S., Oxtoby E., Hughes J. A molecular and biochemical analysis of the structure of the cyanogenic beta-glucosidase (linamarase) from cassava (Manihot esculenta Cranz). Arch Biochem Biophys. 1992 Jun;295(2):273–279. doi: 10.1016/0003-9861(92)90518-2. [DOI] [PubMed] [Google Scholar]
  18. Kaushal G. P., Pastuszak I., Hatanaka K., Elbein A. D. Purification to homogeneity and properties of glucosidase II from mung bean seedlings and suspension-cultured soybean cells. J Biol Chem. 1990 Sep 25;265(27):16271–16279. [PubMed] [Google Scholar]
  19. Kempton J. B., Withers S. G. Mechanism of Agrobacterium beta-glucosidase: kinetic studies. Biochemistry. 1992 Oct 20;31(41):9961–9969. doi: 10.1021/bi00156a015. [DOI] [PubMed] [Google Scholar]
  20. Kengen S. W., Luesink E. J., Stams A. J., Zehnder A. J. Purification and characterization of an extremely thermostable beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus. Eur J Biochem. 1993 Apr 1;213(1):305–312. doi: 10.1111/j.1432-1033.1993.tb17763.x. [DOI] [PubMed] [Google Scholar]
  21. Kiss L., Berki L. K., Nánási P. Evidence for a single catalytic and two binding sites in the almond emulsin beta-D-glucosidase molecule. Biochem Biophys Res Commun. 1981 Feb 12;98(3):792–799. doi: 10.1016/0006-291x(81)91181-5. [DOI] [PubMed] [Google Scholar]
  22. Leah R., Kigel J., Svendsen I., Mundy J. Biochemical and molecular characterization of a barley seed beta-glucosidase. J Biol Chem. 1995 Jun 30;270(26):15789–15797. doi: 10.1074/jbc.270.26.15789. [DOI] [PubMed] [Google Scholar]
  23. Li Y. K., Yao H. J., Pan I. H. Mechanistic study of beta-xylosidase from Trichoderma koningii G-39. J Biochem. 2000 Feb;127(2):315–320. doi: 10.1093/oxfordjournals.jbchem.a022609. [DOI] [PubMed] [Google Scholar]
  24. Ly H. D., Withers S. G. Mutagenesis of glycosidases. Annu Rev Biochem. 1999;68:487–522. doi: 10.1146/annurev.biochem.68.1.487. [DOI] [PubMed] [Google Scholar]
  25. Lymar E. S., Li B., Renganathan V. Purification and Characterization of a Cellulose-Binding (beta)-Glucosidase from Cellulose-Degrading Cultures of Phanerochaete chrysosporium. Appl Environ Microbiol. 1995 Aug;61(8):2976–2980. doi: 10.1128/aem.61.8.2976-2980.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Matsui I., Sakai Y., Matsui E., Kikuchi H., Kawarabayasi Y., Honda K. Novel substrate specificity of a membrane-bound beta-glycosidase from the hyperthermophilic archaeon Pyrococcus horikoshii. FEBS Lett. 2000 Feb 11;467(2-3):195–200. doi: 10.1016/s0014-5793(00)01156-x. [DOI] [PubMed] [Google Scholar]
  27. Namchuk M. N., Withers S. G. Mechanism of Agrobacterium beta-glucosidase: kinetic analysis of the role of noncovalent enzyme/substrate interactions. Biochemistry. 1995 Dec 12;34(49):16194–16202. doi: 10.1021/bi00049a035. [DOI] [PubMed] [Google Scholar]
  28. Pérez-Pons J. A., Rebordosa X., Querol E. Properties of a novel glucose-enhanced beta-glucosidase purified from Streptomyces sp. (ATCC 11238). Biochim Biophys Acta. 1995 Sep 6;1251(2):145–153. doi: 10.1016/0167-4838(95)00074-5. [DOI] [PubMed] [Google Scholar]
  29. Raynal A., Gerbaud C., Francingues M. C., Guerineau M. Sequence and transcription of the beta-glucosidase gene of Kluyveromyces fragilis cloned in Saccharomyces cerevisiae. Curr Genet. 1987;12(3):175–184. doi: 10.1007/BF00436876. [DOI] [PubMed] [Google Scholar]
  30. Sinnott M. L., Souchard I. J. The mechanism of action of beta-galactosidase. Effect of aglycone nature and -deuterium substitution on the hydrolysis of aryl galactosides. Biochem J. 1973 May;133(1):89–98. doi: 10.1042/bj1330089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Street I. P., Kempton J. B., Withers S. G. Inactivation of a beta-glucosidase through the accumulation of a stable 2-deoxy-2-fluoro-alpha-D-glucopyranosyl-enzyme intermediate: a detailed investigation. Biochemistry. 1992 Oct 20;31(41):9970–9978. doi: 10.1021/bi00156a016. [DOI] [PubMed] [Google Scholar]
  32. Varghese J. N., Hrmova M., Fincher G. B. Three-dimensional structure of a barley beta-D-glucan exohydrolase, a family 3 glycosyl hydrolase. Structure. 1999 Feb 15;7(2):179–190. doi: 10.1016/s0969-2126(99)80024-0. [DOI] [PubMed] [Google Scholar]
  33. Wang Q., Trimbur D., Graham R., Warren R. A., Withers S. G. Identification of the acid/base catalyst in Agrobacterium faecalis beta-glucosidase by kinetic analysis of mutants. Biochemistry. 1995 Nov 7;34(44):14554–14562. doi: 10.1021/bi00044a034. [DOI] [PubMed] [Google Scholar]
  34. Watanabe T., Sato T., Yoshioka S., Koshijima T., Kuwahara M. Purification and properties of Aspergillus niger beta-glucosidase. Eur J Biochem. 1992 Oct 15;209(2):651–659. doi: 10.1111/j.1432-1033.1992.tb17332.x. [DOI] [PubMed] [Google Scholar]
  35. Withers S. G., Rupitz K., Trimbur D., Warren R. A. Mechanistic consequences of mutation of the active site nucleophile Glu 358 in Agrobacterium beta-glucosidase. Biochemistry. 1992 Oct 20;31(41):9979–9985. doi: 10.1021/bi00156a017. [DOI] [PubMed] [Google Scholar]

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