From beta-glucanase to beta-glucansynthase: glycosyl transfer to alpha-glycosyl fluorides catalyzed by a mutant endoglucanase lacking its catalytic nucleophile.
Removal of the catalytic nucleophile Glu134 of the retaining 1,3-1,4-beta-glucanase from Bacillus licheniformis by mutation to alanine yields an enzyme with no glycosidase activity. The mutant is able to catalyze the regio- and stereospecific glycosylation of alpha-laminaribiosyl fluoride with different glucoside acceptors through a single-step inverting mechanism. The main advantage of the mutant as glycosylation catalyst with respect to the kinetically controlled transglycosylation using the wild-type enzyme is that the reaction products cannot be hydrolyzed by the mutant enzyme, and glycosylation yields rise to 90%.
Malet
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Laboratory of Biochemistry, Institut Químic de Sarrià, Universitat Ramon Llull, Barcelona, Spain.
Planas
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D019943:Amino Acid Substitution; D001407:Bacillus / Q000201:enzymology*; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid; D005947:Glucose / Q000031:analogs & derivatives* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D012997:Solvents; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
D012997:Solvents; C030364:glucosyl fluoride; D006026:Glycoside Hydrolases; C028020:licheninase; D005947:Glucose