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 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.
Neustroev
Kirill N
KN
Molecular and Radiation Biology Division, Petersburg Nuclear Physics Institute, Russian Academy of Science, Gatchina, 188300, Russia.
Golubev
Alexander M
AM
Sinnott
Michael L
ML
Borriss
Rainer
R
Krah
Martin
M
Brumer
Harry
H
Eneyskaya
Elena V
EV
Shishlyannikov
Sergey
S
Shabalin
Konstantin A
KA
Peshechonov
Viacheslav T
VT
Korolev
Vladimir G
VG
Kulminskaya
Anna A
AA
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
D005936:Glucans; D011134:Polysaccharides; C008247:laminaran; D044602:Cellulases