title: Transferase and hydrolytic activities of the laminarinase from Rhodothermus marinus and its M133A, M133C, and M133W mutants.
abstract: Comparative studies of the transglycosylation and hydrolytic activities have been performed on the Rhodothermus marinus beta-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 beta(1-4) and beta(1-6) linkages. To obtain kinetic probes for the wild-type enzyme and Met-133 mutants, p-nitrophenyl beta-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-beta-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 beta-glucopyranosyl-beta-1,3-D-glucopy- ranosyl-beta-1,3-D-glucopyranose and beta-glucopyranosyl-beta-1, 3-D-glucopyranosyl-beta-1,3-D-glucpyranosyl-beta-1,3-D- glucopyranoxside.
authors.affiliation: Molecular and Radiation Biology Division, Petersburg Nuclear Physics Institute, Russian Academy of Science, Gatchina, 188300, Russia.
authors.forename: Kirill N
authors.initials: KN
authors.lastname: Neustroev
authors.forename: Alexander M
authors.initials: AM
authors.lastname: Golubev
authors.forename: Michael L
authors.initials: ML
authors.lastname: Sinnott
authors.forename: Rainer
authors.initials: R
authors.lastname: Borriss
authors.forename: Martin
authors.initials: M
authors.lastname: Krah
authors.forename: Harry
authors.initials: H
authors.lastname: Brumer
authors.forename: Elena V
authors.initials: EV
authors.lastname: Eneyskaya
authors.forename: Sergey
authors.initials: S
authors.lastname: Shishlyannikov
authors.forename: Konstantin A
authors.initials: KA
authors.lastname: Shabalin
authors.forename: Viacheslav T
authors.initials: VT
authors.lastname: Peshechonov
authors.forename: Vladimir G
authors.initials: VG
authors.lastname: Korolev
authors.forename: Anna A
authors.initials: AA
authors.lastname: Kulminskaya
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002240:Carbohydrate Sequence; D020134:Catalytic Domain / Q000235:genetics; D044602:Cellulases / Q000235:genetics* / Q000378:metabolism*; D005936:Glucans; D006031:Glycosylation; D006868:Hydrolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011134:Polysaccharides / Q000737:chemistry; D045245:Rhodothermus / Q000201:enzymology* / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D005936:Glucans; D011134:Polysaccharides; C008247:laminaran; D044602:Cellulases