An original chemoenzymatic route for the synthesis of β-d-galactofuranosides using an α-l-arabinofuranosidase

Rémond, Caroline; Plantier‐Royon, Richard; Aubry, Nathalie; O’Donohue, Michael

doi:10.1016/j.carres.2005.01.016

Cited by 34 publications

(24 citation statements)

References 47 publications

(35 reference statements)

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“…The same group reported very recently also the synthesis of galactofuranosides using the above catalyst [220].…”

Section: Hardly Used Enzymatic Activitiesmentioning

confidence: 95%

Glycosyl Hydrolases and Glycosyltransferases in the Synthesis of Oligosaccharides

Trincone¹,

Giordano

2006

COC

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Glycobiology and related disciplines have received an enormous interest in recent years as they shed new light on the functional roles of carbohydrates in biological events leading to the understanding of mechanisms of important pathologies and to the development of new therapeutics. Although carbohydrate can be isolated from natural sources, the synthetic strategy plays its own role allowing access to larger quantities of structurally defined material and entry to analogs of naturally occurring structures. Nowadays, the bottleneck in this field is represented by some limitations of the potential of carbohydrate containing molecules because their complex structures make classical chemical synthesis very difficult.The synthesis of oligosaccharides have to face with three main snags: (i) reactivity of the leaving group on the monosaccharide acting as donor; (ii) regioselectivity towards a single hydroxyl group on the acceptor molecule; (iii) stereoselectivity in forming pure anomers of the new glycosidic product. The protecting-group manipulations that are needed for the stereocontrol of the products are demanding procedures thus yields and selectivity are lowered hindering the efficient production of oligosaccharides needed for biological testing.A particular benefit of the renewed interest for sugar chemistry has been the attention of scientific community to the biological methodologies in the production of oligosaccharides, an area which emerged in recent decades.In the carbohydrate field different enzymes were used for diverse purposes but enzymatic strategies for high-yield and stereospecific construction of glycosidic bonds are based on the action of two types of enzymes: glycosyl hydrolases (endo-and exo-glycosidases) and glycosyltransferases. Glycosyl hydrolases in the cells are responsible for the cleavage of glycosidic linkages; the exo-glycosidase are involved for glycan processing during in vivo glycoprotein synthesis. The glycosyltransferases are instead responsible in vivo for the synthesis of most cell-surface glycoconjugates.This review deals with the application of different types of glycosyl hydrolases. Elegant examples concerning the use of genetically modified representatives of glycosyl hydrolases (glycosynthases and thioglycoligases) will be also reported; some recent advances on the use of glycosyltransferases are also included.In compiling this review we were aware of the huge amount of excellent material published in the recent years on this topic, thus we will limit the covering of literature to the last decade. Attention will be devoted to the regioselectivity towards pyranosidic templates and to the yield of reactions. The molecular diversity obtained by the use of enzymes from different biological sources and the biological importance of the compounds synthesized will be focused as well.This review will put in evidence that glycosyl hydrolases and glycosyltransferases for the synthesis of the glycosidic linkages will play a relevant role in the next future as on the bench bio-catalysts ...

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“…The same group reported very recently also the synthesis of galactofuranosides using the above catalyst [220].…”

Section: Hardly Used Enzymatic Activitiesmentioning

confidence: 95%

Glycosyl Hydrolases and Glycosyltransferases in the Synthesis of Oligosaccharides

Trincone¹,

Giordano

2006

COC

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“…18 13 (Table S7) were derived from triplicate experiments. In Figure 6A the lowest LG pKa data point was omitted when establishing the linear data fit (Table S8).…”

Section: -34mentioning

confidence: 99%

“…In several studies, we have shown how, in the presence of appropriate sugar or alcohol acceptors, the H/T partition can be driven towards transglycosylation, allowing the synthesis of a variety of furanose-based compounds, including L-arabinoand D-galactofuranosides. 18,19 Moreover, in recent work 3 using in vitro random mutagenesis we managed to tip the H/T balance further towards transglycosylation, creating novel glycosynthetic tools for the synthesis of complex furanoside-based structures that would be tricky to access using conventional synthetic chemistry. 20,21 In the present study, we have pursued our efforts to engineer TxAbf, this time combining our previously-acquired knowledge to develop a hybrid engineering approach that has relied on random mutagenesis, site-saturation mutagenesis at previously identified hot-spots and sitedirected mutagenesis driven by in silico predictions.…”

Section: Introductionmentioning

confidence: 98%

Molecular Design of Non-Leloir Furanose-Transferring Enzymes from an α-l-Arabinofuranosidase: A Rationale for the Engineering of Evolved Transglycosylases

et al. 2015

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The vast biodiversity of glycoside hydrolases (GHs) constitutes a reservoir of readily-available carbohydrateacting enzymes that employ simple substrates and hold the potential to perform highly stereopecific and regioselective glycosynthetic reactions. However, most GHs preferentially hydrolyze glycosidic bonds and are thus characterized by a hydrolysis/transglycosylation partition in favor of hydrolysis. Unfortunately, current knowledge is insufficient to rationally modify this partition, specifically mutating key molecular determinants to tip the balance towards transglycosylation. In this study, in the absence of precise knowledge concerning the hydrolysis/transglycosylation partition in a hydrolytic GH51 α-L-arabinofuranosidase, we describe how an iterative protein engineering approach has been used to create the first 'non-Leloir' transarabinofuranosylases. In the first step, random mutagenesis yielded a point mutation (R69H) at a position that is highly conserved in clan GH-A. Characterization of R69H revealed that this enzyme displays high transglycosylation activity, but severely reduced (61-fold) activity on pNP-α-L-arabinofuranoside. Upon recombination of R69H with other point mutations selected using semi-rational or in silico approaches, transfer rates close to 100% and transarabinofuranosylation yields of the main (1 2)-linked oligosaccharide product of 80% (vs 11% for the wild-type) were obtained. Combining data presented here with knowledge drawn from the literature, we suggest that the creation of nonLeloir transglycosylases necessarily involves the destabilization of the highly developed transition states that characterize the predominantly hydrolytic exo-acting GHs; this being an efficient way to prevent ubiquitous water molecules from performing the deglycosylation step.

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“…In addition, an α -l -arabinofuranosidase (AbfD3) was found in Thermobacillus xylanilyticus , which displays transglycosylation activity and is able to use pNp-β -Gal f as the sugar donor (Remond et al , 2005 ). These data demonstrate that enzymes exist with affi nity for both Ara f and Gal f and suggest the possibility that one or more arabinofuranosidases in A. niger play a role in the degradation or synthesis of Gal f -containing glycoconjugates.…”

Section: Introductionmentioning

confidence: 75%

Fungal α-arabinofuranosidases of glycosyl hydrolase families 51 and 54 show a dual arabinofuranosyl- and galactofuranosyl-hydrolyzing activity

Tefsen¹,

Lagendijk²,

Park

et al. 2012

Biological Chemistry

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Aspergillus niger possesses a galactofuranosidase activity, however, the corresponding enzyme or gene encoding this enzyme has never been identifi ed. As evidence is mounting that enzymes exist with affi nity for both arabinofuranose and galactofuranose, we investigated the possibility that α -l -arabinofuranosidases, encoded by the abfA and abfB genes, are responsible for the galactofuranosidase activity of A. niger . Characterization of the recombinant AbfA and AbfB proteins revealed that both enzymes do not only hydrolyze p -nitrophenyl-α -l -arabinofuranoside (pNp-α -Ara f ) but are also capable of hydrolyzing p -nitrophenyl-β -d -galactofuranoside (pNp-β -Gal f ). Molecular modeling of the AbfB protein with pNp-β -Gal f confi rmed the possibility for AbfB to interact with this substrate, similarly as with pNp-α -Ara f . We also show that galactomannan, a cell wall compound of A. niger , containing β -linked terminal and internal galactofuranosyl moieties, can be degraded by an enzyme activity that is present in the supernatant of inulin-grown A. niger . Interestingly, purifi ed AbfA and AbfB did not show this hydrolyzing activity toward A. niger galactomannan. In summary, our studies demonstrate that AbfA and AbfB, α -l -arabinofuranosidases from different families, both contain a galactofuranose (Galf) -hydrolyzing activity. In addition, our data support the presence of a Gal fhydrolase activity expressed by A. niger that is capable of degrading fungal galactomannan.

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An original chemoenzymatic route for the synthesis of β-d-galactofuranosides using an α-l-arabinofuranosidase

Cited by 34 publications

References 47 publications

Glycosyl Hydrolases and Glycosyltransferases in the Synthesis of Oligosaccharides

Glycosyl Hydrolases and Glycosyltransferases in the Synthesis of Oligosaccharides

Molecular Design of Non-Leloir Furanose-Transferring Enzymes from an α-l-Arabinofuranosidase: A Rationale for the Engineering of Evolved Transglycosylases

Fungal α-arabinofuranosidases of glycosyl hydrolase families 51 and 54 show a dual arabinofuranosyl- and galactofuranosyl-hydrolyzing activity

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