Hydrolase and glycosynthase activity of endo-1,3-beta-glucanase from the thermophile Pyrococcus furiosus.
Pyrococcus furiosus laminarinase (LamA, PF0076) is an endo-glycosidase that hydrolyzes beta-1,3-glucooligosaccharides, but not beta-1,4-gluco-oligosaccharides. We studied the specificity of LamA towards small saccharides by using 4-methylumbelliferyl beta-glucosides with different linkages. Besides endo-activity, wild-type LamA has some exo-activity, and catalyzes the hydrolysis of mixed-linked oligosaccharides (Glcbeta4Glcbeta3Glcbeta-MU (Glc = glucosyl, MU = 4-methylumbelliferyl)) with both beta-1,4 and beta-1,3 specificities. The LamA mutant E170A had severely reduced hydrolytic activity, which is consistent with Glu170 being the catalytic nucleophile. The E170A mutant was active as a glycosynthase, catalyzing the condensation of alpha-laminaribiosyl fluoride to different acceptors. The best condensation yields were found at pH 6.5 and 50 degrees C, but did not exceed 30%. Depending on the acceptor, the synthase generated either a beta-1,3 or a beta-1,4 linkage.
van Lieshout
J
J
Laboratory for Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Hesselink van Suchtelenweg 4, 6703 CT, Wageningen, The Netherlands. johan.vanlieshout@wur.nl
Faijes
M
M
Nieto
J
J
van der Oost
J
J
Planas
A
A
D019943:Amino Acid Substitution; D020134:Catalytic Domain; D004795:Enzyme Stability; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000378:metabolism*; D005960:Glucosides / Q000378:metabolism; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000378:metabolism*; D006923:Hymecromone / Q000031:analogs & derivatives* / Q000378:metabolism; D020125:Mutation, Missense; D009844:Oligosaccharides / Q000378:metabolism; D020027:Pyrococcus furiosus / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
D005960:Glucosides; D009844:Oligosaccharides; C014110:4-methylumbelliferyl glucoside; D006923:Hymecromone; D006867:Hydrolases; D004693:Glucan Endo-1,3-beta-D-Glucosidase