Glycosynthase activity of Geobacillus stearothermophilus GH52 beta-xylosidase: efficient synthesis of xylooligosaccharides from alpha-D-xylopyranosyl fluoride through a conjugated reaction.
Glycosynthases are mutant glycosidases in which the acidic nucleophile is replaced by a small inert residue. In the presence of glycosyl fluorides of the opposite anomeric configuration (to that of their natural substrates), these enzymes can catalyze glycosidic bond formation with various acceptors. In this study we demonstrate that XynB2E335G, a nucleophile-deficient mutant of a glycoside hydrolase family 52 beta-xylosidase from G. stearothermophilus, can function as an efficient glycosynthase, using alpha-D-xylopyranosyl fluoride as a donor and various aryl sugars as acceptors. The mutant enzyme can also catalyze the self-condensation reaction of alpha-D-xylopyranosyl fluoride, providing mainly alpha-D-xylobiosyl fluoride. The self-condensation kinetics exhibited apparent classical Michaelis-Menten behavior, with kinetic constants of 1.3 s(-1) and 2.2 mM for k(cat) and K(M(acceptor)), respectively, and a k(cat)/K(M(acceptor)) value of 0.59 s(-1) mM(-1). When the beta-xylosidase E335G mutant was combined with a glycoside hydrolase family 10 glycosynthase, high-molecular-weight xylooligomers were readily obtained from the affordable alpha-D-xylopyranosyl fluoride as the sole substrate.
Ben-David
Alon
A
Department of Biotechnology and Food Engineering and Institute of Catalysis Science and Technology, Technion-Israel Institute of Technology, Haifa 32000, Israel.
Bravman
Tsafrir
T
Balazs
Yael S
YS
Czjzek
Mirjam
M
Schomburg
Dietmar
D
Shoham
Gil
G
Shoham
Yuval
Y
D001406:Bacillaceae / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D005964:Glucosyltransferases / Q000378:metabolism*; D007700:Kinetics; D015394:Molecular Structure; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D014994:Xylose / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D014995:Xylosidases / Q000378:metabolism*
D009844:Oligosaccharides; C526946:alpha-D-xylopyranosyl fluoride; D014994:Xylose; D005964:Glucosyltransferases; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase