Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Mar;179(5):1636–1645. doi: 10.1128/jb.179.5.1636-1645.1997

Phospho-beta-glucosidase from Fusobacterium mortiferum: purification, cloning, and inactivation by 6-phosphoglucono-delta-lactone.

J Thompson 1, S A Robrish 1, C L Bouma 1, D I Freedberg 1, J E Folk 1
PMCID: PMC178877  PMID: 9045824

Abstract

6-Phosphoryl-beta-D-glucopyranosyl:6-phosphoglucohydrolase (P-beta-glucosidase, EC 3.2.1.86) has been purified from Fusobacterium mortiferum. Assays for enzyme activity and results from Western immunoblots showed that P-beta-glucosidase (Mr, 53,000; pI, 4.5) was induced by growth of F. mortiferum on beta-glucosides. The novel chromogenic and fluorogenic substrates, p-nitrophenyl-beta-D-glucopyranoside-6-phosphate (pNPbetaGlc6P) and 4-methylumbelliferyl-beta-D-glucopyranoside-6-phosphate (4MUbetaGlc6P), respectively, were used for the assay of P-beta-glucosidase activity. The enzyme hydrolyzed several P-beta-glucosides, including the isomeric disaccharide phosphates cellobiose-6-phosphate, gentiobiose-6-phosphate, sophorose-6-phosphate, and laminaribiose-6-phosphate, to yield glucose-6-phosphate and appropriate aglycons. The kinetic parameters for each substrate are reported. P-beta-glucosidase from F. mortiferum was inactivated by 6-phosphoglucono-delta-lactone (P-glucono-delta-lactone) derived via oxidation of glucose 6-phosphate. The pbgA gene that encodes P-beta-glucosidase from F. mortiferum has been cloned and sequenced. The first 42 residues deduced from the nucleotide sequence matched those determined for the N terminus by automated Edman degradation of the purified enzyme. From the predicted sequence of 466 amino acids, two catalytically important glutamyl residues have been identified. Comparative alignment of the amino acid sequences of P-beta-glucosidase from Escherichia coli and F. mortiferum indicates potential binding sites for the inhibitory P-glucono-delta-lactone to the enzyme from F. mortiferum.

Full Text

The Full Text of this article is available as a PDF (740.7 KB).

Selected References

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

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Baker P. J., Britton K. L., Rice D. W., Rob A., Stillman T. J. Structural consequences of sequence patterns in the fingerprint region of the nucleotide binding fold. Implications for nucleotide specificity. J Mol Biol. 1992 Nov 20;228(2):662–671. doi: 10.1016/0022-2836(92)90848-e. [DOI] [PubMed] [Google Scholar]
  3. Beutler E., Kuhl W. Characteristics and significance of the reverse glucose-6-phosphate dehydrogenase reaction. J Lab Clin Med. 1986 Jun;107(6):502–507. [PubMed] [Google Scholar]
  4. Brendel V., Trifonov E. N. A computer algorithm for testing potential prokaryotic terminators. Nucleic Acids Res. 1984 May 25;12(10):4411–4427. doi: 10.1093/nar/12.10.4411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. González-Candelas L., Ramón D., Polaina J. Sequences and homology analysis of two genes encoding beta-glucosidases from Bacillus polymyxa. Gene. 1990 Oct 30;95(1):31–38. doi: 10.1016/0378-1119(90)90410-s. [DOI] [PubMed] [Google Scholar]
  6. Gräbnitz F., Seiss M., Rücknagel K. P., Staudenbauer W. L. Structure of the beta-glucosidase gene bglA of Clostridium thermocellum. Sequence analysis reveals a superfamily of cellulases and beta-glycosidases including human lactase/phlorizin hydrolase. Eur J Biochem. 1991 Sep 1;200(2):301–309. doi: 10.1111/j.1432-1033.1991.tb16186.x. [DOI] [PubMed] [Google Scholar]
  7. Hall B. G., Xu L. Nucleotide sequence, function, activation, and evolution of the cryptic asc operon of Escherichia coli K12. Mol Biol Evol. 1992 Jul;9(4):688–706. doi: 10.1093/oxfordjournals.molbev.a040753. [DOI] [PubMed] [Google Scholar]
  8. Hengstenberg W., Kohlbrecher D., Witt E., Kruse R., Christiansen I., Peters D., Pogge von Strandmann R., Städtler P., Koch B., Kalbitzer H. R. Structure and function of proteins of the phosphotransferase system and of 6-phospho-beta-glycosidases in gram-positive bacteria. FEMS Microbiol Rev. 1993 Sep;12(1-3):149–163. doi: 10.1111/j.1574-6976.1993.tb00016.x. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Henrissat B., Callebaut I., Fabrega S., Lehn P., Mornon J. P., Davies G. Conserved catalytic machinery and the prediction of a common fold for several families of glycosyl hydrolases. Proc Natl Acad Sci U S A. 1995 Jul 18;92(15):7090–7094. doi: 10.1073/pnas.92.15.7090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. KUNDIG W., GHOSH S., ROSEMAN S. PHOSPHATE BOUND TO HISTIDINE IN A PROTEIN AS AN INTERMEDIATE IN A NOVEL PHOSPHO-TRANSFERASE SYSTEM. Proc Natl Acad Sci U S A. 1964 Oct;52:1067–1074. doi: 10.1073/pnas.52.4.1067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. LEABACK D. H., WALKER P. G. Studies on glucosaminidase. 4. The fluorimetric assay of N-acetyl-beta-glucosaminidase. Biochem J. 1961 Jan;78:151–156. doi: 10.1042/bj0780151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lalégerie P., Legler G., Yon J. M. The use of inhibitors in the study of glycosidases. Biochimie. 1982 Nov-Dec;64(11-12):977–1000. doi: 10.1016/s0300-9084(82)80379-9. [DOI] [PubMed] [Google Scholar]
  14. Leaback D. H. On the inhibition of beta-N-acetyl-D-glucosaminidase by 2-acetamido-2-deoxy-D-glucono-(1-5)-lactone. Biochem Biophys Res Commun. 1968 Sep 30;32(6):1025–1030. doi: 10.1016/0006-291x(68)90132-0. [DOI] [PubMed] [Google Scholar]
  15. Legler G. Glycoside hydrolases: mechanistic information from studies with reversible and irreversible inhibitors. Adv Carbohydr Chem Biochem. 1990;48:319–384. doi: 10.1016/s0065-2318(08)60034-7. [DOI] [PubMed] [Google Scholar]
  16. Levvy G. A., Hay A. J., Conchie J. Inhibition of glycosidases by aldonolactones of corresponding configuration. 4. Inhibitors of mannosidase and glucosidase. Biochem J. 1964 May;91(2):378–384. doi: 10.1042/bj0910378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Meadow N. D., Fox D. K., Roseman S. The bacterial phosphoenolpyruvate: glycose phosphotransferase system. Annu Rev Biochem. 1990;59:497–542. doi: 10.1146/annurev.bi.59.070190.002433. [DOI] [PubMed] [Google Scholar]
  18. Palmer R. E., Anderson R. L. Cellobiose metabolism in Aerobacter aerogenes. 3. Cleavage of cellobiose monophosphate by a phospho- -glucosidase. J Biol Chem. 1972 Jun 10;247(11):3420–3423. [PubMed] [Google Scholar]
  19. Palmer R. E., Anderson R. L. Cellobiose metabolism in Aerobacter aerogenes. II. Phosphorylation of cellobiose with adenosine 5'-triphosphate by a -glucoside kinase. J Biol Chem. 1972 Jun 10;247(11):3415–3419. [PubMed] [Google Scholar]
  20. Parker L. L., Hall B. G. Characterization and nucleotide sequence of the cryptic cel operon of Escherichia coli K12. Genetics. 1990 Mar;124(3):455–471. doi: 10.1093/genetics/124.3.455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Postma P. W., Lengeler J. W., Jacobson G. R. Phosphoenolpyruvate:carbohydrate phosphotransferase systems of bacteria. Microbiol Rev. 1993 Sep;57(3):543–594. doi: 10.1128/mr.57.3.543-594.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Reizer J., Saier M. H., Jr, Deutscher J., Grenier F., Thompson J., Hengstenberg W. The phosphoenolpyruvate:sugar phosphotransferase system in gram-positive bacteria: properties, mechanism, and regulation. Crit Rev Microbiol. 1988;15(4):297–338. doi: 10.3109/10408418809104461. [DOI] [PubMed] [Google Scholar]
  24. Robrish S. A., Fales H. M., Gentry-Weeks C., Thompson J. Phosphoenolpyruvate-dependent maltose:phosphotransferase activity in Fusobacterium mortiferum ATCC 25557: specificity, inducibility, and product analysis. J Bacteriol. 1994 Jun;176(11):3250–3256. doi: 10.1128/jb.176.11.3250-3256.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Robrish S. A., Oliver C., Thompson J. Sugar metabolism by fusobacteria: regulation of transport, phosphorylation, and polymer formation by Fusobacterium mortiferum ATCC 25557. Infect Immun. 1991 Dec;59(12):4547–4554. doi: 10.1128/iai.59.12.4547-4554.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Robrish S. A., Thompson J. Regulation of fructose metabolism and polymer synthesis by Fusobacterium nucleatum ATCC 10953. J Bacteriol. 1990 Oct;172(10):5714–5723. doi: 10.1128/jb.172.10.5714-5723.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rojas A., Arola L., Romeu A. beta-Glucosidase families revealed by computer analysis of protein sequences. Biochem Mol Biol Int. 1995 May;35(6):1223–1231. [PubMed] [Google Scholar]
  28. Roseman S. Sialic acid, serendipity, and sugar transport: discovery of the bacterial phosphotransferase system. FEMS Microbiol Rev. 1989 Jun;5(1-2):3–11. doi: 10.1016/0168-6445(89)90003-x. [DOI] [PubMed] [Google Scholar]
  29. Saier M. H., Jr, Reizer J. Proposed uniform nomenclature for the proteins and protein domains of the bacterial phosphoenolpyruvate: sugar phosphotransferase system. J Bacteriol. 1992 Mar;174(5):1433–1438. doi: 10.1128/jb.174.5.1433-1438.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schnetz K., Toloczyki C., Rak B. Beta-glucoside (bgl) operon of Escherichia coli K-12: nucleotide sequence, genetic organization, and possible evolutionary relationship to regulatory components of two Bacillus subtilis genes. J Bacteriol. 1987 Jun;169(6):2579–2590. doi: 10.1128/jb.169.6.2579-2590.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Schofield P. J., Sols A. Rat liver 6-phosphogluconolactonase: a low Km enzyme. Biochem Biophys Res Commun. 1976 Aug 23;71(4):1313–1318. doi: 10.1016/0006-291x(76)90798-1. [DOI] [PubMed] [Google Scholar]
  32. Scrutton N. S., Berry A., Perham R. N. Redesign of the coenzyme specificity of a dehydrogenase by protein engineering. Nature. 1990 Jan 4;343(6253):38–43. doi: 10.1038/343038a0. [DOI] [PubMed] [Google Scholar]
  33. Tanaka A., Ito M., Hiromi K. Equilibrium and kinetic studies on the binding of gluconolactone to almond beta-glucosidase in the absence and presence of glucose. J Biochem. 1986 Nov;100(5):1379–1385. doi: 10.1093/oxfordjournals.jbchem.a121844. [DOI] [PubMed] [Google Scholar]
  34. Thompson J., Gentry-Weeks C. R., Nguyen N. Y., Folk J. E., Robrish S. A. Purification from Fusobacterium mortiferum ATCC 25557 of a 6-phosphoryl-O-alpha-D-glucopyranosyl:6-phosphoglucohydrolase that hydrolyzes maltose 6-phosphate and related phospho-alpha-D-glucosides. J Bacteriol. 1995 May;177(9):2505–2512. doi: 10.1128/jb.177.9.2505-2512.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Thompson J., Nguyen N. Y., Robrish S. A. Sucrose fermentation by Fusobacterium mortiferum ATCC 25557: transport, catabolism, and products. J Bacteriol. 1992 May;174(10):3227–3235. doi: 10.1128/jb.174.10.3227-3235.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wierenga R. K., Terpstra P., Hol W. G. Prediction of the occurrence of the ADP-binding beta alpha beta-fold in proteins, using an amino acid sequence fingerprint. J Mol Biol. 1986 Jan 5;187(1):101–107. doi: 10.1016/0022-2836(86)90409-2. [DOI] [PubMed] [Google Scholar]
  37. Wiesmann C., Beste G., Hengstenberg W., Schulz G. E. The three-dimensional structure of 6-phospho-beta-galactosidase from Lactococcus lactis. Structure. 1995 Sep 15;3(9):961–968. doi: 10.1016/s0969-2126(01)00230-1. [DOI] [PubMed] [Google Scholar]
  38. Wilson G., Fox C. F. The beta-glucoside system of Escherichia coli. IV. Purification and properties of phospho-beta-glucosidases A and B. J Biol Chem. 1974 Sep 10;249(17):5586–5598. [PubMed] [Google Scholar]
  39. 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]
  40. 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]
  41. Zhang J., Aronson A. A Bacillus subtilis bglA gene encoding phospho-beta-glucosidase is inducible and closely linked to a NADH dehydrogenase-encoding gene. Gene. 1994 Mar 11;140(1):85–90. doi: 10.1016/0378-1119(94)90735-8. [DOI] [PubMed] [Google Scholar]
  42. el Hassouni M., Henrissat B., Chippaux M., Barras F. Nucleotide sequences of the arb genes, which control beta-glucoside utilization in Erwinia chrysanthemi: comparison with the Escherichia coli bgl operon and evidence for a new beta-glycohydrolase family including enzymes from eubacteria, archeabacteria, and humans. J Bacteriol. 1992 Feb;174(3):765–777. doi: 10.1128/jb.174.3.765-777.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES