Characterization of Glycosynthase Mutants Derived from Glycoside Hydrolase Family 10 Xylanases

Sugimura, Masahiro; Nishimoto, Mamoru; Kitaoka, Motomitsu

doi:10.1271/bbb.70.1210

Cited by 37 publications

(24 citation statements)

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“…[17] Very recently, it was demonstrated that GH10 xylanases can serve as glycosynthases and produce xylooligosaccharides when a-xylobiosyl fluoride is used as a donor. [18,19] Here we report the first glycosynthase A C H T U N G T R E N N U N G derived from a b-xylosidase-the GH52 b-xylosidase from G. stearothermophilus. In addition to being an effective glycosynthase, this enzyme can be combined with a GH10 xylanase mutant to produce xylooligomers from a-d-xylopyranosyl fluoride as the sole substrate.…”

Section: Introductionmentioning

confidence: 92%

“…The mutant enzyme was found to catalyze the formation of aryl-oligosaccharides ranging in length from tetra-to dodecasaccharides. In another study, Sugimura et al [19] reported the generation of glycosynthases from GH10 xylanases originating from four different bacterial sources. These enzymes were able to produce polymeric b-(1!4)-linked xylopyranoside.…”

Section: Synthesis Of Xylooligosaccharides In a Conjugated Reactionmentioning

confidence: 99%

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

et al. 2007

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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.

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Section: Introductionmentioning

confidence: 92%

Section: Synthesis Of Xylooligosaccharides In a Conjugated Reactionmentioning

confidence: 99%

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

et al. 2007

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“…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].…”

Section: Artificial Polysaccharides By Glycosynthasesmentioning

confidence: 99%

When Enzymes Do It Better: Enzymatic Glycosylation Methods

Planas¹,

Faijes²,

Codera³

2015

Carbohydrate Chemistry: State of the Art and Challenges for Drug Development

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Glycoside Bond Formation by CAZymesCarbohydrates, in the forms of oligosaccharides, polysaccharides, and glycoconjugates (glycoproteins, glycolipids, glycosylated metabolites, etc) are key biomolecules in all living organisms. They play roles in a variety of biological functions as structural components of cell walls, reserves of stored energy, and mediators of cell-cell interactions, being central components in many important biological recognition processes, such as cell adhesion and signaling, cancer progression, host-pathogen interactions, and immune responses [1][2][3][4][5].Synthetic tools to access natural and non-natural glycosides and glycoconjugates have become a central issue in glycochemistry and glycobiology. Both for industrial applications in diverse fields, such as medicine, pharmacology, food, cosmetics, etc., as well as for biological research in functional glycomic studies, there is the need for efficient synthetic approaches for structurally defined oligosaccharides and glycoconjugates. In contrast to conventional chemical synthesis that requires complex and tedious protection/deprotection manipulations and activation strategies to achieve regio-and Carbohydrate Chemistry: State of the Art and Challenges for Drug Development Downloaded from www.worldscientific.com by UNIVERSITY OF PITTSBURGH on 10/02/15. For personal use only.

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“…Concept to obtain glycosynthase activity from an inverting GH The relationship between the F -releasing activity and the hydrolytic activity of Rex during acquiring the glycosynthase activity was completely diŠerent from those in retaining enzymes. In the case of retaining enzymes, the parent enzyme does not possess F -releasing activity (15,41), and a mutation at the base residue completely eliminates the hydrolytic activity because the base residue directly attacks the carbon 1 (C1) of the hydrolyzed glycoside. Thus, while converting retaining GHs into glycosynthases, it is important to acquire F -releasing activity through mutations.…”

Section: E Conversion Of Rex Into Glycosynthase E-1 Hehre Resynthesmentioning

confidence: 99%

Conversion of inverting glycoside hydrolases into catalysts for synthesizing glycosides employing a glycosynthase strategy

Kitaoka

Honda

Fushinobu

et al. 2009

TIGG

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23Trends in Glycoscience and Glycotechnology Vol. 21 No. 117 (2009) AbstractReducing-end xylose-releasing exo-oligoxylanase (Rex, EC. 3.2.1.156) is an inverting xylanolytic enzyme, belonging to the glycoside hydrolase (GH) family 8, which hydrolyzes xylooligosaccharides to release xylose (X1) from its reducing end. Rex hydrolyzes a-xylobiosyl ‰uo-ride (a-X2F) to yield xylobiose (X2) only in the presence of X1, conˆrming the Hehre resynthesis-hydrolysis mechanism. A library of mutant Rex at the catalytic base (D263) was constructed by saturation mutagenesis, in which D263C accumulated the highest level of xylotriose (X 3 ) from a-X 2 F and X 1 . However, F -releasing activities of the mutants were much less than that of the wild type. Next, Y198 residue of Rex that forms a hydrogen bond with nucleophilic water was substituted with phenylalanine, causing a marked decrease in hydrolytic activity and a small increase in the F -releasing activity from a-X2F in the presence of X1. Y198F of Rex accumulated more product during the glycosynthase reaction than D263C. Recently, an inverting a-1,2-fucosidase belonging to GH95 was converted into glycosynthase by mutating a catalytic base residue. In both cases, the catalytic base should be intact.

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Characterization of Glycosynthase Mutants Derived from Glycoside Hydrolase Family 10 Xylanases

Cited by 37 publications

References 51 publications

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

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

When Enzymes Do It Better: Enzymatic Glycosylation Methods

Conversion of inverting glycoside hydrolases into catalysts for synthesizing glycosides employing a glycosynthase strategy

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