Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 Dec;177(24):7105–7111. doi: 10.1128/jb.177.24.7105-7111.1995

Characterization of the celB gene coding for beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus and its expression and site-directed mutation in Escherichia coli.

W G Voorhorst 1, R I Eggen 1, E J Luesink 1, W M de Vos 1
PMCID: PMC177588  PMID: 8522516

Abstract

The celB gene encoding the cellobiose-hydrolyzing enzyme beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus has been identified, cloned, and sequenced. The transcription and translation gene was overexpressed in Escherichia coli, resulting in high-level (up to 20% of total protein) production of beta-glucosidase that could be purified by a two-step purification procedure. The beta-glucosidase produced by E. coli had kinetic and stability properties similar to those of the beta-glucosidase purified from P. furiosus. The deduced amino acid sequence of CelB showed high similarity with those of beta-glycosidases that belong to glycosyl hydrolase family 1, implicating a conserved structure. Replacement of the conserved glutamate 372 in the P. furiosus beta-glucosidase by an aspartate or a glutamine led to a high reduction in specific activity (200- or 1,000-fold, respectively), indicating that this residue is the active site nucleophile involved in catalysis above 100 degrees C.

Full Text

The Full Text of this article is available as a PDF (326.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Brown J. W., Daniels C. J., Reeve J. N. Gene structure, organization, and expression in archaebacteria. Crit Rev Microbiol. 1989;16(4):287–338. doi: 10.3109/10408418909105479. [DOI] [PubMed] [Google Scholar]
  2. Casadaban M. J., Chou J., Cohen S. N. In vitro gene fusions that join an enzymatically active beta-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J Bacteriol. 1980 Aug;143(2):971–980. doi: 10.1128/jb.143.2.971-980.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  4. Costantino H. R., Brown S. H., Kelly R. M. Purification and characterization of an alpha-glucosidase from a hyperthermophilic archaebacterium, Pyrococcus furiosus, exhibiting a temperature optimum of 105 to 115 degrees C. J Bacteriol. 1990 Jul;172(7):3654–3660. doi: 10.1128/jb.172.7.3654-3660.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. DiRuggiero J., Robb F. T., Jagus R., Klump H. H., Borges K. M., Kessel M., Mai X., Adams M. W. Characterization, cloning, and in vitro expression of the extremely thermostable glutamate dehydrogenase from the hyperthermophilic Archaeon, ES4. J Biol Chem. 1993 Aug 25;268(24):17767–17774. [PubMed] [Google Scholar]
  7. Diruggiero J., Robb F. T. Expression and in vitro assembly of recombinant glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus. Appl Environ Microbiol. 1995 Jan;61(1):159–164. doi: 10.1128/aem.61.1.159-164.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Eggen R. I., Geerling A. C., Jetten M. S., de Vos W. M. Cloning, expression, and sequence analysis of the genes for carbon monoxide dehydrogenase of Methanothrix soehngenii. J Biol Chem. 1991 Apr 15;266(11):6883–6887. [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. 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]
  12. Kengen S. W., de Bok F. A., van Loo N. D., Dijkema C., Stams A. J., de Vos W. M. Evidence for the operation of a novel Embden-Meyerhof pathway that involves ADP-dependent kinases during sugar fermentation by Pyrococcus furiosus. J Biol Chem. 1994 Jul 1;269(26):17537–17541. [PubMed] [Google Scholar]
  13. Laderman K. A., Asada K., Uemori T., Mukai H., Taguchi Y., Kato I., Anfinsen C. B. Alpha-amylase from the hyperthermophilic archaebacterium Pyrococcus furiosus. Cloning and sequencing of the gene and expression in Escherichia coli. J Biol Chem. 1993 Nov 15;268(32):24402–24407. [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Maras B., Valiante S., Chiaraluce R., Consalvi V., Politi L., De Rosa M., Bossa F., Scandurra R., Barra D. The amino acid sequence of glutamate dehydrogenase from Pyrococcus furiosus, a hyperthermophilic archaebacterium. J Protein Chem. 1994 Feb;13(2):253–259. doi: 10.1007/BF01891983. [DOI] [PubMed] [Google Scholar]
  16. Mukund S., Adams M. W. The novel tungsten-iron-sulfur protein of the hyperthermophilic archaebacterium, Pyrococcus furiosus, is an aldehyde ferredoxin oxidoreductase. Evidence for its participation in a unique glycolytic pathway. J Biol Chem. 1991 Aug 5;266(22):14208–14216. [PubMed] [Google Scholar]
  17. Prisco A., Moracci M., Rossi M., Ciaramella M. A gene encoding a putative membrane protein homologous to the major facilitator superfamily of transporters maps upstream of the beta-glycosidase gene in the archaeon Sulfolobus solfataricus. J Bacteriol. 1995 Mar;177(6):1614–1619. doi: 10.1128/jb.177.6.1614-1619.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Rowlands T., Baumann P., Jackson S. P. The TATA-binding protein: a general transcription factor in eukaryotes and archaebacteria. Science. 1994 May 27;264(5163):1326–1329. doi: 10.1126/science.8191287. [DOI] [PubMed] [Google Scholar]
  19. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  21. Uemori T., Ishino Y., Toh H., Asada K., Kato I. Organization and nucleotide sequence of the DNA polymerase gene from the archaeon Pyrococcus furiosus. Nucleic Acids Res. 1993 Jan 25;21(2):259–265. doi: 10.1093/nar/21.2.259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Wettach J., Gohl H. P., Tschochner H., Thomm M. Functional interaction of yeast and human TATA-binding proteins with an archaeal RNA polymerase and promoter. Proc Natl Acad Sci U S A. 1995 Jan 17;92(2):472–476. doi: 10.1073/pnas.92.2.472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Witt E., Frank R., Hengstenberg W. 6-Phospho-beta-galactosidases of gram-positive and 6-phospho-beta-glucosidase B of gram-negative bacteria: comparison of structure and function by kinetic and immunological methods and mutagenesis of the lacG gene of Staphylococcus aureus. Protein Eng. 1993 Nov;6(8):913–920. doi: 10.1093/protein/6.8.913. [DOI] [PubMed] [Google Scholar]
  25. Zwickl P., Fabry S., Bogedain C., Haas A., Hensel R. Glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus woesei: characterization of the enzyme, cloning and sequencing of the gene, and expression in Escherichia coli. J Bacteriol. 1990 Aug;172(8):4329–4338. doi: 10.1128/jb.172.8.4329-4338.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES