Article

Glycoconjugate Journal

, Volume 23, Issue 7, pp 501-511

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

  • Kirill N. NeustroevAffiliated withMolecular and Radiation Biology Division, Petersburg Nuclear Physics Institute, Russian Academy of Science
  • , Alexander M. GolubevAffiliated withMolecular and Radiation Biology Division, Petersburg Nuclear Physics Institute, Russian Academy of Science
  • , Michael L. SinnottAffiliated withDepartment of Chemical and Biological Sciences, University of Huddersfield, Queensgate
  • , Rainer BorrissAffiliated withAG Bakteriengenetik, Institut fur Biologie, Humboldt Universitt Berlin Chausseestrasse 117
  • , Martin KrahAffiliated withAG Bakteriengenetik, Institut fur Biologie, Humboldt Universitt Berlin Chausseestrasse 117
  • , Harry BrumerIIIAffiliated withDepartment of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Centre
  • , Elena V. EneyskayaAffiliated withMolecular and Radiation Biology Division, Petersburg Nuclear Physics Institute, Russian Academy of Science
  • , Sergey ShishlyannikovAffiliated withMolecular and Radiation Biology Division, Petersburg Nuclear Physics Institute, Russian Academy of Science
  • , Konstantin A. ShabalinAffiliated withMolecular and Radiation Biology Division, Petersburg Nuclear Physics Institute, Russian Academy of Science
    • , Viacheslav T. PeshechonovAffiliated withMolecular and Radiation Biology Division, Petersburg Nuclear Physics Institute, Russian Academy of Science
    • , Vladimir G. KorolevAffiliated withMolecular and Radiation Biology Division, Petersburg Nuclear Physics Institute, Russian Academy of Science
    • , Anna A. KulminskayaAffiliated withMolecular and Radiation Biology Division, Petersburg Nuclear Physics Institute, Russian Academy of Science Email author 

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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, k cat/K m, 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