1887

Abstract

strain 121 uses sucrose for synthesis of a unique, soluble glucan (‘reuteran’) with mainly -(1→4) glucosidic linkages. The gene () encoding this glucansucrase enzyme had previously been characterized. Here, a detailed biochemical and molecular analysis of the GTFA enzyme is presented. This is believed to be the first report describing reuteransucrase enzyme kinetics and the oligosaccharides synthesized with various acceptors. Alignments of the GTFA sequence with glucansucrases from and identified conserved amino-acid residues in the catalytic core critical for enzyme activity. Mutants Asp1024Asn, Glu1061Gln and Asp1133Asn displayed 300- to 1000-fold-reduced specific activities. To investigate the role of the relatively large N-terminal variable domain (702 amino acids) and the relatively short C-terminal putative glucan-binding domain (267 amino acids, with 11 YG repeats), various truncated derivatives of GTFA (1781 amino acids) were constructed and characterized. Deletion of the complete N-terminal variable domain of GTFA (GTFA-ΔN) had little effect on reuteran characteristics (size, distribution of glycosidic linkages), but the initial transferase activity of the mutant enzyme increased drastically. Sequential C-terminal deletions (up to six YG repeats) in GTFA-ΔN also had little effect on reuteran characteristics. However, enzyme kinetics drastically changed. Deletion of 7, 8 or 11 YG repeats resulted in dramatic loss of total enzyme activity (43-, 63- and 1000-fold-reduced specific activities, respectively). Characterization of sequential C-terminal deletion mutants of GTFA-ΔN revealed that the C-terminal domain of reuteransucrase has an important role in glucan binding.

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2004-07-01
2019-12-09
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References

  1. Abo, H., Matsumura, T., Kodama, T., Ohta, H., Fukui, K., Kato, K. & Kagawa, H. ( 1991; ). Peptide sequences for sucrose splitting and glucan binding within Streptococcus sobrinus glucosyltransferase (water-insoluble glucan synthetase). J Bacteriol 173, 989–996.
    [Google Scholar]
  2. Argüello-Morales, M. A., Remaud-Simeon, M., Pizzut, S., Sarçabal, P., Willemot R.-M & Monsan, P. ( 2000; ). Sequence analysis of the gene encoding alternansucrase, a sucrose glucosyltransferase from Leuconostoc mesenteroides NRRL B-1355. FEMS Microbiol Lett 182, 81–85.[CrossRef]
    [Google Scholar]
  3. Argüello Morales, M. A., Remaud-Simeon, M., Willemot, R.-M., Vignon, M. R. & Monsan, P. ( 2001; ). Novel oligosaccharides synthesized from sucrose donor and cellobiose acceptor by alternansucrase. Carbohydr Res 331, 403–411.[CrossRef]
    [Google Scholar]
  4. Ausubel, F. M., Brent, R. E., Kingston, D. D., Moore, J. G., Seidman, J. G., Smith, J. A. & Struhl, K. ( 1987; ). Current Protocols in Molecular Biology. New York: Wiley.
  5. Bozonnet, S., Dols-Laffargue, M., Fabre, E., Pizzut, S., Remaud-Simeon, M., Monsan, P. & Willemot, R.-M. ( 2002; ). Molecular characterization of DSR-E, an α-1,2 linkage-synthesizing dextransucrase with two catalytic domains. J Bacteriol 184, 5753–5761.[CrossRef]
    [Google Scholar]
  6. Buchholz, K. & Seibel, J. ( 2003; ). Isomaltooligosaccharides. In Oligosaccharides in Food and Agriculture, pp. 63–75. Edited by G. Eggleston & G. L. Côté. Washington: American Chemical Society.
  7. Chaplin, M. F. ( 1982; ). A rapid and sensitive method for the analysis of carbohydrate components in glycoproteins using gas-liquid chromatography. Anal Biochem 123, 336–341.[CrossRef]
    [Google Scholar]
  8. Cote, G. L. & Robyt, J. F. ( 1982; ). Isolation and partial characterization of an extracellular glucansucrase from Leuconostoc mesenteroides NRRL B-1355 that synthesizes an alternating (1→6), (1→3)-α-d-glucan. Carbohydr Res 101, 57–74.[CrossRef]
    [Google Scholar]
  9. Devulapalle, K. S., Goodman, S. D., Gao, Q., Hemsley, A. & Mooser, G. ( 1997; ). Knowledge-based model of a glucosyltransferase from the oral bacterial group of mutans streptococci. Protein Sci 6, 2489–2493.
    [Google Scholar]
  10. Dols, M., Simeon, M. R., Willemot, R. M., Vignon, M. R. & Monsan, P. F. ( 1997; ). Structural characterization of the maltose acceptor-products synthesized by Leuconostoc mesenteroides NRRL B-1299 dextransucrase. Carbohydr Res 305, 549–559.[CrossRef]
    [Google Scholar]
  11. Ferretti, J. J., Gilpin, M. L. & Russell, R. R. ( 1987; ). Nucleotide sequence of a glucosyltransferase gene from Streptococcus sobrinus MFe28. J Bacteriol 169, 4271–4278.
    [Google Scholar]
  12. Funane, K., Mizuno, K., Takahara, H. & Kobayashi, M. ( 2000; ). Gene encoding a dextransucrase-like protein in Leuconostoc mesenteroides NRRL B-512F. Biosci Biotechnol Biochem 64, 29–38.[CrossRef]
    [Google Scholar]
  13. Giffard, P. M. & Jacques, N. A. ( 1994; ). Definition of a fundamental repeating unit in streptococcal glucosyltransferase glucan-binding regions and related sequences. J Dent Res 73, 1133–1141.
    [Google Scholar]
  14. Giffard, P. M., Simpson, C. L., Milward, C. P. & Jacques, N. A. ( 1991; ). Molecular characterization of a cluster of at least two glucosyltransferase genes in Streptococcus salivarius ATCC 25975. J Gen Microbiol 137, 2577–2593.[CrossRef]
    [Google Scholar]
  15. Giffard, P. M., Allen, D. M., Milward, C. P., Simpson, C. L. & Jacques, N. A. ( 1993; ). Sequence of the gtfK gene of Streptococcus salivarius ATCC 25975 and evolution of the gtf genes of oral streptococci. J Gen Microbiol 139, 1511–1522.[CrossRef]
    [Google Scholar]
  16. Gilmore, K. S., Russell, R. R. & Ferretti, J. J. ( 1990; ). Analysis of the Streptococcus downei gtfS gene, which specifies a glucosyltransferase that synthesizes soluble glucans. Infect Immun 58, 2452–2458.
    [Google Scholar]
  17. Hakomori, S. ( 1964; ). A rapid permethylation of glycolipid and polysaccharide catalyzed by methylsulfinyl carbanion in dimethyl sulfoxide. J Biochem 55, 205–208.
    [Google Scholar]
  18. Hanahan, D. ( 1983; ). Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166, 557–580.[CrossRef]
    [Google Scholar]
  19. Harris, P. J., Henry, R. J., Blakeney, A. B. & Stone, B. A. ( 1984; ). An improved procedure for the methylation analysis of oligosaccharides and polysaccharides. Carbohydr Res 127, 59–73.[CrossRef]
    [Google Scholar]
  20. Honda, O., Kato, C. & Kuramitsu, H. K. ( 1990; ). Nucleotide sequence of the Streptococcus mutans gtfD gene encoding the glucosyltransferase-S enzyme. J Gen Microbiol 136, 2099–2105.[CrossRef]
    [Google Scholar]
  21. Janecek, S., Svensson, B. & Russell, R. R. ( 2000; ). Location of repeat elements in glucansucrases of Leuconostoc and Streptococcus species. FEMS Microbiol Lett 192, 53–57.[CrossRef]
    [Google Scholar]
  22. Jansson, P.-E., Kenne, L., Liedgren, H., Lindberg, B. & Lönngren, J. ( 1976; ). A practical guide to the methylation analysis of carbohydrates. Chem Commun Univ Stockholm 8, 1–74.
    [Google Scholar]
  23. Kato, C. & Kuramitsu, H. K. ( 1990; ). Carboxyl-terminal deletion analysis of the Streptococcus mutans glucosyltransferase-I enzyme. FEMS Microbiol Lett 72, 299–302.[CrossRef]
    [Google Scholar]
  24. Kato, C., Nakano, Y., Lis, M. & Kuramitsu, H. K. ( 1992; ). Molecular genetic analysis of the catalytic site of Streptococcus mutans glucosyltransferases. Biochem Biophys Res Commun 189, 1184–1188.[CrossRef]
    [Google Scholar]
  25. Kralj, S., van Geel-Schutten, G. H., Rahaoui, H., Leer, R. J., Faber, E. J., van der Maarel, M. J. & Dijkhuizen, L. ( 2002; ). Molecular characterization of a novel glucosyltransferase from Lactobacillus reuteri strain 121 synthesizing a unique, highly branched glucan with α-(1→4) and α-(1→6) glucosidic bonds. Appl Environ Microbiol 68, 4283–4291.[CrossRef]
    [Google Scholar]
  26. Laemmli, U. K. ( 1970; ). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef]
    [Google Scholar]
  27. Lis, M., Shiroza, T. & Kuramitsu, H. K. ( 1995; ). Role of C-terminal direct repeating units of the Streptococcus mutans glucosyltransferase-S in glucan binding. Appl Environ Microbiol 61, 2040–2042.
    [Google Scholar]
  28. MacGregor, E. A., Jespersen, H. M. & Svensson, B. ( 1996; ). A circularly permuted α-amylase-type α/β-barrel structure in glucan-synthesizing glucosyltransferases. FEBS Lett 378, 263–266.[CrossRef]
    [Google Scholar]
  29. McCarter, J. D. & Withers, S. G. ( 1994; ). Mechanisms of enzymatic glycoside hydrolysis. Curr Opin Struct Biol 4, 885–892.[CrossRef]
    [Google Scholar]
  30. Monchois, V., Willemot, R. M., Remaud-Simeon, M., Croux, C. & Monsan, P. ( 1996; ). Cloning and sequencing of a gene coding for a novel dextransucrase from Leuconostoc mesenteroides NRRL B-1299 synthesizing only α(1-6) and α(1-3) linkages. Gene 182, 23–32.[CrossRef]
    [Google Scholar]
  31. Monchois, V., Remaud-Simeon, M., Russell, R. R., Monsan, P. & Willemot, R. M. ( 1997; ). Characterization of Leuconostoc mesenteroides NRRL B-512F dextransucrase (DSRS) and identification of amino-acid residues playing a key role in enzyme activity. Appl Microbiol Biotechnol 48, 465–472.[CrossRef]
    [Google Scholar]
  32. Monchois, V., Remaud-Simeon, M., Monsan, P. & Willemot, R. M. ( 1998a; ). Cloning and sequencing of a gene coding for an extracellular dextransucrase (DSRB) from Leuconostoc mesenteroides NRRL B-1299 synthesizing only a α(1-6) glucan. FEMS Microbiol Lett 159, 307–315.
    [Google Scholar]
  33. Monchois, V., Reverte, A., Remaud-Simeon, M., Monsan, P. & Willemot, R. M. ( 1998b; ). Effect of Leuconostoc mesenteroides NRRL B-512F dextransucrase carboxy-terminal deletions on dextran and oligosaccharide synthesis. Appl Environ Microbiol 64, 1644–1649.
    [Google Scholar]
  34. Monchois, V., Arguello-Morales, M. & Russell, R. R. B. ( 1999a; ). Isolation of an active catalytic core of Streptococcus downei MFe28 GTF-I glucosyltransferase. J Bacteriol 181, 2290–2292.
    [Google Scholar]
  35. Monchois, V., Vignon, M. & Russell, R. R. ( 1999b; ). Isolation of key amino acid residues at the N-terminal end of the core region Streptococcus downei glucansucrase, GTF-I. Appl Microbiol Biotechnol 52, 660–665.[CrossRef]
    [Google Scholar]
  36. Monchois, V., Willemot, R. M. & Monsan, P. ( 1999c; ). Glucansucrases: mechanism of action and structure-function relationships. FEMS Microbiol Rev 23, 131–151.
    [Google Scholar]
  37. Monchois, V., Vignon, M., Escalier, P. C., Svensson, B. & Russell, R. R. ( 2000a; ). Involvement of Gln937 of Streptococcus downei GTF-I glucansucrase in transition-state stabilization. Eur J Biochem 267, 4127–4136.[CrossRef]
    [Google Scholar]
  38. Monchois, V., Vignon, M. & Russell, R. R. ( 2000b; ). Mutagenesis of Asp-569 of glucosyltransferase I glucansucrase modulates glucan and oligosaccharide synthesis. Appl Environ Microbiol 66, 1923–1927.[CrossRef]
    [Google Scholar]
  39. Mooser, G., Hefta, S. A., Paxton, R. J., Shively, J. E. & Lee, T. D. ( 1991; ). Isolation and sequence of an active-site peptide containing a catalytic aspartic acid from two Streptococcus sobrinus α-glucosyltransferases. J Biol Chem 266, 8916–8922.
    [Google Scholar]
  40. Mukasa, H., Shimamura, A. & Tsumori, H. ( 2000; ). Nigerooligosaccharide acceptor reaction of Streptococcus sobrinus glucosyltransferase GTF-I. Carbohydr Res 326, 98–103.[CrossRef]
    [Google Scholar]
  41. Murray, V. ( 1989; ). Improved double-stranded DNA sequencing using the linear polymerase chain reaction. Nucleic Acids Res 17, 8889.[CrossRef]
    [Google Scholar]
  42. Potocki de Montalk, G., Remaud-Simeon, M., Willemot, R. M., Sarcabal, P., Planchot, V. & Monsan, P. ( 2000; ). Amylosucrase from Neisseria polysaccharea: novel catalytic properties. FEBS Lett 471, 219–223.[CrossRef]
    [Google Scholar]
  43. Robyt, J. F. ( 1996; ). Mechanism and action of glucansucrase. In Enzymes for Carbohydrate Engineering, pp. 1–22. Edited by K. H. Park, J. F. Robyt & Y. D. Choi. Amsterdam: Elsevier Science.
  44. Robyt, J. F. & Walseth, T. F. ( 1978; ). The mechanism of acceptor reactions of Leuconostoc mesenteroides B-512F dextransucrase. Carbohydr Res 61, 433–445.[CrossRef]
    [Google Scholar]
  45. Sambrook, J., Fritsch, E. F. & Maniates, T. ( 1989; ). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  46. Sarkar, G. & Sommer, S. S. ( 1990; ). The ‘megaprimer’ method of site-directed mutagenesis. Biotechniques 8, 404–407.
    [Google Scholar]
  47. Shah, D. S. H. & Russell, R. R. ( 2002; ). Glucan binding domain of streptococcal glucosyltransferases. Biologia 57, 129–136.
    [Google Scholar]
  48. Shiroza, T., Ueda, S. & Kuramitsu, H. K. ( 1987; ). Sequence analysis of the gtfB gene from Streptococcus mutans. J Bacteriol 169, 4263–4270.
    [Google Scholar]
  49. Uitdehaag, J. C., Mosi, R., Kalk, K. H., van der Veen, B. A., Dijkhuizen, L., Withers, S. G. & Dijkstra, B. W. ( 1999; ). X-ray structures along the reaction pathway of cyclodextrin glycosyltransferase elucidate catalysis in the alpha-amylase family. Nat Struct Biol 6, 432–436.[CrossRef]
    [Google Scholar]
  50. van der Veen, B. A., Uitdehaag, J. C., Dijkstra, B. W. & Dijkhuizen, L. ( 2000; ). Engineering of cyclodextrin glycosyltransferase reaction and product specificity. Biochim Biophys Acta 1543, 336–360.[CrossRef]
    [Google Scholar]
  51. van Geel-Schutten, G. H., Faber, E. J., Smit, E., Bonting, K., Smith, M. R., Ten Brink, B., Kamerling, J. P., Vliegenthart, J. F. & Dijkhuizen, L. ( 1999; ). Biochemical and structural characterization of the glucan and fructan exopolysaccharides synthesized by the Lactobacillus reuteri wild-type strain and by mutant strains. Appl Environ Microbiol 65, 3008–3014.
    [Google Scholar]
  52. van Hijum, S. A., van Geel-Schutten, G. H., Rahaoui, H., van der Maarel, M. J. & Dijkhuizen, L. ( 2002; ). Characterization of a novel fructosyltransferase from Lactobacillus reuteri that synthesizes high-molecular-weight inulin and inulin oligosaccharides. Appl Environ Microbiol 68, 4390–4398.[CrossRef]
    [Google Scholar]
  53. van Hijum, S. A., van der Maarel, M. J. & Dijkhuizen, L. ( 2003; ). Kinetic properties of an inulosucrase from Lactobacillus reuteri 121. FEBS Lett 534, 207–210.[CrossRef]
    [Google Scholar]
  54. van Hijum, S. A. F. T., Szalowska, E., van der Maarel, M. J. E. C. & Dijkhuizen, L. ( 2004; ). Biochemical and molecular characterization of a levansucrase from Lactobacillus reuteri. Microbiology 150, 621–630.[CrossRef]
    [Google Scholar]
  55. Vickerman, M. M., Sulavik, M. C., Minick, P. E. & Clewell, D. B. ( 1996; ). Changes in the carboxyl-terminal repeat region affect extracellular activity and glucan products of Streptococcus gordonii glucosyltransferase. Infect Immun 64, 5117–5128.
    [Google Scholar]
  56. Wunder, D. & Bowen, W. H. ( 1999; ). Action of agents on glucosyltransferases from Streptococcus mutans in solution and adsorbed to experimental pellicle. Arch Oral Biol 44, 203–214.[CrossRef]
    [Google Scholar]
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