Glycosynthase Activity of Geobacillus stearothermophilus GH52 β‐Xylosidase: Efficient Synthesis of Xylooligosaccharides from α‐D‐Xylopyranosyl Fluoride through a Conjugated Reaction

Abstract

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.

A New Family of Carbohydrate Esterases Is Represented by a GDSL Hydrolase/Acetylxylan Esterase from Geobacillus stearothermophilus

“…The bacterium degrades xylan by initially secreting an extracellular xylanase (7)(8)(9)(10), which partially degrades xylan to decorated xylo-oligomers that are transported into the cell via a specific ATP-binding cassette (ABC) transport system (11). Inside the cell, the decorated xylo-oligomers are hydrolyzed by side chain-cleaving enzymes, arabinofuranosidases (12)(13)(14) and glucuronidase (15)(16)(17), and finally by intracellular xylanase (18) and xylosidases (19,20).…”

When Enzymes Do It Better: Enzymatic Glycosylation Methods

“…Endo-xylanases from the GH10 family have been converted to glycosynthases that polymerize α-xylobiosyl fluoride to afford polymeric β-1,4-xylan with DP ≈ 30 [105], as well as from GH52 β-xylosidase [106].…”

Glycosidases in Carbohydrate Synthesis: When Organic Chemistry Falls Short

“…[41] Glycosylations with 6-O-sulfo-N-acetyl-d-glucosaminyl moiety was demonstrated in several recent works. [45] Loft and Williams [46] published challenging syntheses of regioselectively sulfated p-nitrophenyl 2-acetomido-2deoxy-b-d-glucopyranosides (6-O-sulfate, cosynthase [65] mutants with altered substrate specificity have been obtained. An example is the xylosynthase derived from Agrobacterium sp.…”