α-Glucosidase Mutant Catalyzes “α-Glycosynthase”-type Reaction

Okuyama, Masayuki; Mori, Haruhide; Watanabe, Kotomi; Kimura, Atsuo; Chiba, Seiya

doi:10.1271/bbb.66.928

Cited by 78 publications

(50 citation statements)

References 23 publications

(37 reference statements)

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“…Thus the carboxyl group as proton donor is essential for the initial action of the mutant. Similar things are suggested for mutants of retaining -glucosidase 80) and -glucanase. 79,88) Recently, mutant enzymes of an interesting -1,4-xylanase releasing from the reducing end of -1,4-oligosaccharide have been reported.…”

Section: )supporting

confidence: 70%

“…80) The actions of the two mutants E134A 79) and D481G 80) also were confirmed to convert from retaining to inverting mode, and the mutants catalyzed the synthesis of oligosaccharide derivatives, that lacked the activity of hydrolysis. The former E134A utilized -laminaribiosyl fluoride and -glucoside (or -cellobioside and laminaribioside), and the latter D481G utilized -glucosyl fluoride and -glucoside.…”

Section: Reaction Mechanism Of Mutant Glycosidasementioning

confidence: 99%

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A Historical Perspective for the Catalytic Reaction Mechanism of Glycosidase; So As to Bring about Breakthrough in Confusing Situation

Chiba

2012

Bioscience, Biotechnology, and Biochemistry

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Various catalytic reaction models have been proposed as the reaction mechanisms of glycosidases, but a reasonable and unitary model capable of interpreting both ''inverting'' and ''retaining'' glycosidase reactions remains to be established. As for the models proposed to date, the nucleophilic displacement mechanism and the oxocarbenium ion intermediate mechanism are widely known, but recently the former is widely accepted, and so the general tendency of world opinion appears to favor it. This reaction model, however, is considered to comprise some inconsistencies that cannot be neglected from the viewpoint of reactivity in organic chemistry. While the nucleophilic displacement mechanism is often applied to reactions of glycosidases, it appears unlikely that such reactions actually occur. This review argues that the oxocarbenium ion intermediate reaction mechanism is more rational than the nucleophilic displacement reaction mechanism, as the action mode of glycosidases and related enzymes.

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Section: )supporting

confidence: 70%

Section: Reaction Mechanism Of Mutant Glycosidasementioning

confidence: 99%

A Historical Perspective for the Catalytic Reaction Mechanism of Glycosidase; So As to Bring about Breakthrough in Confusing Situation

Chiba

2012

Bioscience, Biotechnology, and Biochemistry

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“…The persistent sugar peak remained without any decrease in peak-area 1 after a 96-h treatment with β-glucosidase, showing that the mixture contains a β-glucosidase- 2 insensitive oligosaccharide. The enzyme certainly hydrolyzes β-Glc-(1→4)-β-Glc linkages, and 3 the results suggest that the mixture contains non-reducing α-D-Galp(1↔1)β-D-Glcp-(4←1)β-D- 4 Glcp resulting from binding of the reducing and non-reducing ends of glucose moieties of 5 acceptor cellobiose at subsites +1 and +2, respectively. The unhydrolyzed oligosaccharide was 6 subjected to methylation analysis.…”

mentioning

confidence: 94%

“…4B). 4 A single trisaccharide species (ESI-MS: m/z 527.16 for C18H32O16 + Na + ) was produced 5 when lactose was used as the acceptor molecule. The NMR spectra indicated that the trisaccharide 6 was β-lactosyl α-D-galactopyranoside [α-D-Galp-(1↔1)-β-D-Glcp-(4←1)β-D-Galp; Gal-Lac], 7 which respective occupancies of β-glucosyl and β-galactosyl moieties at subsites +1 and +2 8 formed.…”

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confidence: 99%

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Efficient synthesis of α‐galactosyl oligosaccharides using a mutant Bacteroides thetaiotaomicron retaining α‐galactosidase (BtGH97b)

et al. 2017

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Abbreviations: α-GalF, α-galactopyranosyl fluoride; Gal-Lac, β-lactosyl α-D-galactopyranoside; 20 GH, glycoside hydrolase family; HMBC, heteronuclear multiple bond correlation; 21 high-performance anion exchange chromatography-pulsed amperometric detection. The preparation of a glycosynthase, a catalytic nucleophile mutant of a glycosidase, is a well-5 established strategy for the effective synthesis of glycosidic linkages. However, glycosynthases 6 derived from α-glycosidases can give poor yields of desired products because they require 7 generally unstable β-glycosyl fluoride donors. Here, we investigate a transglycosylation catalyzed 8 by a catalytic nucleophile mutant derived from a glycoside hydrolase family (GH) 97 α-9 galactosidase, using more stable β-galactosyl azide and α-galactosyl fluoride donors. The mutant 10 enzyme catalyzes the glycosynthase reaction using β-galactosyl azide and α-galactosyl transfer 11 from α-galactosyl fluoride with assistance of external anions. Formate was more effective at 12 restoring transfer activity than azide. Kinetic analysis suggests that poor transglycosylation in the 13 presence of the azide is because of low activity of the ternary complex between enzyme, β-14 galactosyl azide, and acceptor. A three-dimensional structure of the mutant enzyme in complex 15 with the transglycosylation product, β-lactosyl α-D-galactoside, was solved to elucidate the 16 ligand-binding aspects of the α-galactosidase. Subtle differences at the β→α loops 1, 2, and 3 of 17 the catalytic TIM barrel of the α-galactosidase from those of a homologous GH97 α-glucoside 18 hydrolase seem to be involved in substrate recognitions. In particular, the Trp residues in β→α 19 loop 1 have separate roles. Trp312 of the α-galactosidase appears to exclude the equatorial 20 hydroxy group at C4 of glucosides, whereas the corresponding Trp residue in the α-glucoside 21 hydrolase makes a hydrogen bond with this hydroxy group. The mechanism of α-galactoside-22 recognition is conserved among GH27, 31, 36, and 97 α-galactosidases. 23 24

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Glycosynthases from Inverting Hydrolases

Kitaoka

2010

Biocatalysis and Biomolecular Engineering

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α-Glucosidase Mutant Catalyzes “α-Glycosynthase”-type Reaction

Cited by 78 publications

References 23 publications

A Historical Perspective for the Catalytic Reaction Mechanism of Glycosidase; So As to Bring about Breakthrough in Confusing Situation

A Historical Perspective for the Catalytic Reaction Mechanism of Glycosidase; So As to Bring about Breakthrough in Confusing Situation

Efficient synthesis of α‐galactosyl oligosaccharides using a mutant Bacteroides thetaiotaomicron retaining α‐galactosidase (BtGH97b)

Glycosynthases from Inverting Hydrolases

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