Glycoconjugate Journal

, Volume 23, Issue 7–8, pp 501–511 | Cite as

Transferase and hydrolytic activities of the laminarinase from rhodothermus marinus and its M133A, M133C, and M133W mutants

  • Kirill N. Neustroev
  • Alexander M. Golubev
  • Michael L. Sinnott
  • Rainer Borriss
  • Martin Krah
  • Harry BrumerIII
  • Elena V. Eneyskaya
  • Sergey Shishlyannikov
  • Konstantin A. Shabalin
  • Viacheslav T. Peshechonov
  • Vladimir G. Korolev
  • Anna A. Kulminskaya
Article

Abstract

Comparative studies of the transglycosylation and hydrolytic activities have been performed on the Rhodothermus marinus β-1,3-glucanase (laminarinase) and its M133A, M133C, and M133W mutants. The M133C mutant demonstrated near 20% greater rate of transglycosylation activity in comparison with the M133A and M133W mutants that was measured by NMR quantitation of nascent β(1-4) and β(1-6) linkages. To obtain kinetic probes for the wild-type enzyme and Met-133 mutants, p-nitrophenyl β-laminarin oligosaccharides of degree of polymerisation 2–8 were synthesized enzymatically. Catalytic efficiency values, kcat/Km, of the laminarinase catalysed hydrolysis of these oligosaccharides suggested possibility of four negative and at least three positive binding subsites in the active site. Comparison of action patterns of the wild-type and M133C mutant in the hydrolysis of the p-nitrophenyl-β-D-oligosac- charides indicated that the increased transglycosylation activity of the M133C mutant did not result from altered subsite affinities. The stereospecificity of the transglycosylation reaction also was unchanged in all mutants; the major transglycosylation products in hydrolysis of p-nitrophenyl laminaribioside were β-glucopyranosyl-β-1,3-D-glucopy- ranosyl-β-1,3-D-glucopyranose and β-glucopyranosyl-β-1, 3-D-glucopyranosyl-β-1,3-D-glucpyranosyl-β-1,3-D- glucopyranoxside.

Keywords

Laminarinase Rhodothermus marinus p-nitrophenyl β-laminarin oligosaccharides Transglycosylation 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Henrissat, B.: A classification of glycosyl hydrolases based on amino acid sequence similarities, Biochem. J. 280, 309–16 (1991)PubMedGoogle Scholar
  2. 2.
    Michel, C., Chantalat, L., Duee, E., Barbeyron, T., Henrissat, B., Kloareg, B., Dideberg, O.: The κ-carrageenanase of P. carrageenovora features a tunnel-shaped active site: a novel insight in the evolution of clan B glycoside hydrolases. Structure 9, 513–25 (2001)PubMedCrossRefGoogle Scholar
  3. 3.
    Planas, A.: Bacterial 1,3-1,4-β-glucanases: structure, function and protein engineering. Biochim. Biophys. Acta 1543, 361–82 (2000)PubMedGoogle Scholar
  4. 4.
    Allouch, J., Jam, M., Helbert, W., Barbeyron, T., Kloareg, B., Henrissat, B., Czjzek, M.: The three-dimensional structures of two β-agarases. J. Biol. Chem. 278, 47171–80 (2003)PubMedCrossRefGoogle Scholar
  5. 5.
    Johansson, P., Brumer, H., Baumann, M.J., Kalla, A.M., Henriksson, H., Denman, S.E., Teeri, T.T., Jones, T.A.: Crystal Structures of poplar xyloglucan endotransglycosylase reveal details of transglycosylation acceptor binding. Plant. Cell. 16, 874–86 (2004)PubMedCrossRefGoogle Scholar
  6. 6.
    Vocadlo, D.J., Davies, G.J., Laine, R., Withers, S.G.: catalysis by hen egg-white lysozyme proceeds via a cavalent intermediate. Nature 412, 835–8 (2001)PubMedCrossRefGoogle Scholar
  7. 7.
    Sinnott, M.L., Catalytic mechanisms of enzymatic glycosyl transfer. Chem. Rev. 90, 1171–1202 (1990)Google Scholar
  8. 8.
    Krah, M., Misselwitz, R., Politz, O., Thomsen, K.K., Welfle, H., Borriss, R.: The laminarinase from thermophilic eubacterium Rhodothermus marinus. Conformation, stability, and identification of active site carboxylic residues by site-directed mutagenesis. Eur. J. Biochem. 257, 101–11 (1998)Google Scholar
  9. 9.
    Godfrey, T.: On comparison of key characteristics of industrial enzymes by type and source. In: Godfrey, T., Reinchelt, J.(eds.) Industrial Enzymology pp. 466. Macmillan, London (1983)Google Scholar
  10. 10.
    Jakeman, D., Withers, S.G., Glycosynthases: new tools for oligosaccharide synthesis. Trends Glycosc. Glycotechnol. 14, 13–25 (2002)Google Scholar
  11. 11.
    Mackenzie, L.F., Wang, Q., Warren, R.A., Withers, S.G., Glycosynthases: mutant glycosidases for oligosaccharide synthesis. J. Am. Chem. Soc. 120, 5583–4 (1998)CrossRefGoogle Scholar
  12. 12.
    Mayer, C., Zechel, D.L., Reid, S.R., Warren, A.J., Withers, S.G.: The E358S mutant of Agrobacterium sp. β-glucosidase is a greatly improved glycosynthase. FEBS Let. 466, 40–4 (2000)Google Scholar
  13. 13.
    Viladot, J.-L., Canals, F., Batllori, X., Planas, A.: Long-lived glycosyl-enzyme intermediate mimic produced by formate re-activation of a mutant endoglucanase lacking its catalytic nucleophile. Biochem. J. 355, 79–86 (2001)PubMedCrossRefGoogle Scholar
  14. 14.
    Williams, S.J., Withers, S.: Glycosyl fluorides in enzymatic reactions. Carbohydr. Res. 327, 27–46 (2000)PubMedCrossRefGoogle Scholar
  15. 15.
    Rivera, M.H., Lypez-Munguha, A., Soberyn, X., Saab-Rincyn, G.: α-Amylase from Bacillus licheniformis mutants near to the catalytic site: effects on hydrolytic and transglycosylation activity. Protein Engineer 16, 505–14 (2003)CrossRefGoogle Scholar
  16. 16.
    Matsui, I., Yoneda, S., Ishikawa, K., Miyairi, S., Fukui, S., Umeyama, H., Honda, K.: Roles of the aromatic residues conserved in the active center of Saccharomycopsis α-amylase for transglycosylation and hydrolysis activity. Biochem. 33, 451–8 (1994)CrossRefGoogle Scholar
  17. 17.
    Hansson, T., Kaper, T., van der Oost, J., de Vos, W.M., Aldercreutz, P.: Improved oligosaccharide synthesis by protein engineering of β-glucosidase CelB from hyperthermophilic Pyrococcus furiosus. Biotechnol. Bioengin. 73, 203–10 (2001)CrossRefGoogle Scholar
  18. 18.
    Viladot, J.-L., Stone, B., Driguez, H., Planas, A.: Expeditious synthesis of a new hexasaccharide using transglycosylation reaction catalysed by Bacillus (13),(14)-β-D-glucan 4-glucanohydrolase. Carbohydr. Res. 311, 95–9 (1998)PubMedCrossRefGoogle Scholar
  19. 19.
    Viladot, J.-L., Moreau, V., Planas, A., Driguez, H.: Transglycosylation activity of Bacillus 1,3-1,4-β-D-glucan 4-glucanohydrolase. Enzymatic studies of alternate 1,3-1,4-β-D-glucooligosaccharides. J. Chem. Soc., Perkin Trans 1, 2383–7 (1997)CrossRefGoogle Scholar
  20. 20.
    Borriss, R., Krah, M., Brumer 3rd, H., Kerzhner, M.A., Elyakova, L.A., Ivanen, D.R., Eneyskaya, E.V., Shishlyannikov, S.M., Shabalin, K.A., Neustroev, K.N.: Enzymatic synthesis of 4-methylumbelliferyl β-(1,3)-D-glucooligosaccharides–new substrates for 1,3(4)-β-glucanase. Carbohydr. Res. 338, 1455–7 (2003)PubMedCrossRefGoogle Scholar
  21. 21.
    Kataoka, K., Muta, T., Yamazaki, S., Takeshige, K.: Activation of macrophages by linear (13)-β-D-glucans. Implications for the recognition of fungi by innate immunity. J. Biol. Chem. 277, 36825–31 (2002)PubMedCrossRefGoogle Scholar
  22. 22.
    Lowe, E., Rice, P., Ha T, Li C, Kelley, J., Ensley, H., Lopez-Perez, J., Kalbfleisch, J., Lowman, D., Margl, P., Browder, W.D., Williams, A.: (1,3)-β-D-linked heptasaccharide is the unit ligand for glucan pattern recognition receptors on human monocytes. Microbes. Infec. 3, 789–97 (2001)CrossRefGoogle Scholar
  23. 23.
    Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–5 (1970)PubMedCrossRefGoogle Scholar
  24. 24.
    Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J.: Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193, 265–75 (1951)PubMedGoogle Scholar
  25. 25.
    Ogawa, K., Tsurugi, J., Watanabe, T.: The dependence of the conformation of a (1,3)-β-D-glucan on chain-length in alkaline solution. Carbohydr. Res. 29, 397–403 (1973)CrossRefGoogle Scholar
  26. 26.
    Petersen, B.O., Krah, M., Duus, J.O., Thomsen, K.K.: A transglycosylating 1,3(4)-β-glucanase from Rhodothermus marinus NMR analysis of enzyme reactions. Eur. J. Biochem. 267, 361–9 (2000)PubMedCrossRefGoogle Scholar
  27. 27.
    Kulminskaya, A.A., Thomsen, K.K., Shabalin, K.A., Sidorenko, I.A., Eneyskaya, E.V., Savel’ev, A.N., Neustroev, K.N.: Isolation, enzymatic properties, and mode of action of an exo-1,3-β-glucanase from Trichoderma viride. Eur. J. Biochem. 268, 6123–31 (2001)Google Scholar
  28. 28.
    Somogyi, M.: Notes on sugar determination. J. Biol. Chem. 195, 19–23 (1952)Google Scholar
  29. 29.
    Eneyskaya, E.V., Brumer 3rd H., Backinowsky, L.V., Ivanen, D.R., Kulminskaya, A.A., Shabalin, K.A., Neustroev, K.N.: Enzymatic synthesis of β-xylanase substrates: Transglycosylation reactions of the β-xylosidase from Aspergillus sp., Carbohydr. Res. 338, 313–25 (2003)Google Scholar
  30. 30.
    Malet, C., Planas, A.: Mechanism of Bacillus 1,3;1,4-β-D-glucan 4-glucanohydrolases: kinetics and pH studies with 4-methylumbelliferyl β-D-glucan oligosaccharides. Biochem. 36, 13838–48 (1997)CrossRefGoogle Scholar
  31. 31.
    Fujimoto, H., Isomura, M., Miyazaki, T., Matsuo, I., Walton, R., Sakakibara, T., Ajisaka, K.: Enzymatic syntheses of GlcNAc β-1-2Man and Gal-β-1-4GlcNAc β-1-2Man as components of complex type sugar chains. Glycoconj. J. 14, 75–80 (1997)PubMedCrossRefGoogle Scholar
  32. 32.
    Pitson, S.M., Seviour, R.J., McDougall, B.M., Woodward, J.R., Stone, B.A.: Purification and characterization of three extracellular (13)-β-D-glucan glucohydrolases from the filamentous fungus Acremonium persicinum. Biochem. J. 308, 733–41 (1995)Google Scholar
  33. 33.
    Kraulis, P.J.: MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallog. 24, 946–50 (1991)CrossRefGoogle Scholar
  34. 34.
    Christensen, U., Olsen, K., Stoffer, B.B., Svensson, B.: Substrate binding mechanism of Glu180 − > Gln, Asp176 - > Asn and wild-type glucoamylases from Aspergillus niger. Biochem. 35, 15009–18 (1996)Google Scholar
  35. 35.
    Christensen, T., Stoffer, B.B., Svensson, B., Christensen, U.: Some details of the reaction mechanism of glucoamylase from Aspergillus niger - Kinetic and structural studies on Trp52 − > Phe and Trp 317 − > Phe mutants. Eur. J. Biochem. 250, 638–45 (1997)PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science + Business Media, LLC 2006

Authors and Affiliations

  • Kirill N. Neustroev
    • 1
  • Alexander M. Golubev
    • 1
  • Michael L. Sinnott
    • 2
  • Rainer Borriss
    • 3
  • Martin Krah
    • 3
  • Harry BrumerIII
    • 4
  • Elena V. Eneyskaya
    • 1
  • Sergey Shishlyannikov
    • 1
  • Konstantin A. Shabalin
    • 1
  • Viacheslav T. Peshechonov
    • 1
  • Vladimir G. Korolev
    • 1
  • Anna A. Kulminskaya
    • 1
  1. 1.Molecular and Radiation Biology DivisionPetersburg Nuclear Physics Institute, Russian Academy of ScienceGatchinaRussia
  2. 2.Department of Chemical and Biological SciencesUniversity of Huddersfield, QueensgateHuddersfieldUK
  3. 3.AG Bakteriengenetik, Institut fur BiologieHumboldt Universitt Berlin Chausseestrasse 117BerlinGermany
  4. 4.Department of Biotechnology, Royal Institute of Technology (KTH)AlbaNova University CentreStockholmSweden

Personalised recommendations