Transferase and hydrolytic activities of the laminarinase from rhodothermus marinus and its M133A, M133C, and M133W mutants
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.
KeywordsLaminarinase Rhodothermus marinus p-nitrophenyl β-laminarin oligosaccharides Transglycosylation
Unable to display preview. Download preview PDF.
- 7.Sinnott, M.L., Catalytic mechanisms of enzymatic glycosyl transfer. Chem. Rev. 90, 1171–1202 (1990)Google Scholar
- 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.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.Jakeman, D., Withers, S.G., Glycosynthases: new tools for oligosaccharide synthesis. Trends Glycosc. Glycotechnol. 14, 13–25 (2002)Google Scholar
- 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
- 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
- 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
- 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.Somogyi, M.: Notes on sugar determination. J. Biol. Chem. 195, 19–23 (1952)Google Scholar
- 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
- 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
- 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