Specificity studies of bacillus 1,3-1,4-beta-glucanases and application to glycosynthase-catalyzed transglycosylation.
Bacillus 1,3-1,4-beta-glucanases hydrolyze 1,3-1,4-beta-gluco-oligosaccharides with a retaining mechanism. The binding-site cleft of these endoglycosidases is composed of six subsites (-4 to +2) of which subsite -3 makes the largest contribution to transition state stabilization. The specificity of this subsite is here analyzed for both glycosidase and glycosynthase activities in the wild-type and the nucleophile-less E134A mutant Bacillus licheniformis enzymes. A D-galactosyl residue on the nonreducing end of a trisaccharide substrate is accepted by the enzyme and binds at subsite -3 in the productive enzyme-substrate complex. The wild-type enzyme catalyzes the hydrolysis of the substrate Glcbeta4Glcbeta3GlcbetaMU (Glc=glucosyl, MU=4-methylumbelliferyl) with a k(cat)/K(M) value only 1.3-fold higher than for the Galbeta4Glcbeta3GlcbetaMU (Gal=galactosyl) substrate. The corresponding alpha-fluorides act as good donors for the glycosynthase condensation reaction with mono- and disaccharide acceptors catalyzed by the E134A mutant. Whereas self-condensation and elongation products are also obtained as minor compounds with the Glcbeta4Glcbeta3GlcalphaF donor, nearly quantitative yields of single condensation products are obtained with the Galbeta4Glcbeta3GlcalphaF donor, in which the axial configuration of the 4-OH group on the nonreducing end prevents self-condensation and elongation reactions.
Fairweather
Jon K
JK
Centre de Recherche sur les Macromolécules Végétales, CNRS, Université J. Fourier, BP 53, 38041 Grenoble Cedex 9, France.
Faijes
Magda
M
Driguez
Hugues
H
Planas
Antoni
A
D019943:Amino Acid Substitution; D001407:Bacillus / Q000201:enzymology*; D002384:Catalysis; D020134:Catalytic Domain; D044644:Endo-1,3(4)-beta-Glucanase; D005461:Fluorine; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006031:Glycosylation; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D015202:Protein Engineering; D013379:Substrate Specificity; D014312:Trisaccharides / Q000378:metabolism
D014312:Trisaccharides; D005461:Fluorine; D006026:Glycoside Hydrolases; D044644:Endo-1,3(4)-beta-Glucanase