Highly Efficient Synthesis of β(1 → 4)-Oligo- and -Polysaccharides Using a Mutant Cellulase

Fort, Sébastien; Boyer, Viviane; Greffe, Lionel; Davies, G.J.; Moroz, Olga V.; Christiansen, Lars; Schülein, M.; Cottaz, Sylvain; Driguez, Hugues

doi:10.1021/ja9936520

Cited by 142 publications

(99 citation statements)

References 40 publications

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“…Using this mutant E231G (1→3)-␤-D-glucan glycosynthase, thiolaminaribiosyl fluoride was condensed with 4NPGlc to generate the trisaccharide 4NP-G3SG3OG ( Figure 3A) in 50% yield. The condensation reaction with thiolaminaribiosyl fluoride was less efficient than for other syntheses using glycosynthases, for which yields of 80 to 100% have been reported Fort et al, 2000). Nevertheless, NMR analysis of the product clearly indicated that the newly formed linkage was in the ␤-anomeric configuration ( 1 H ␦ 4.45, J 7.5 Hz) and joined to C3 of the D-glucopyranosyl unit bearing the aromatic 4NP aglycone ( 13 C ␦ 86.3) (Bock et al, 1984).…”

Section: Synthesis Of 4-np S-(␤-d-glucopyranosyl)-(1→3)-(3-thio-␤-d-gmentioning

confidence: 89%

“…The mixture was separated by chromatography on a reverse-phase C 18 silica gel column using a methanol/ water gradient (0 to 20%) to yield, after evaporation and lyophiliza- (Fort et al, 2000;Driguez, 2001). …”

Section: Preparation Of 4-np S-(␤-d-glucopyranosyl)-(1→3)-(3-thio-mentioning

confidence: 99%

“…A nonhydrolyzable (1→4)-␤-linked substrate analog 4 I ,4 III ,4 V -S-trithiocellohexaose (G4SG4OG4SG4OG4SG) was synthesized previously (Driguez, 2001) and was used to examine the S-cellobioside/enzyme complex . Figure 3A) was synthesized using "glycosynthase" methodology Malet and Planas, 1998;Fort et al, 2000). A mutant barley (1→3)-␤-D-glucan endohydrolase isoenzyme GII (E231G), in which the catalytic nucleophile has been altered to a Gly residue, acts as a highly efficient glycosynthase for the generation of (1→3)-␤-D-glucan polymers .…”

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Structural Basis for Broad Substrate Specificity in Higher Plant β-d-Glucan Glucohydrolases

et al. 2002

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Family 3 ␤ -D -glucan glucohydrolases are distributed widely in higher plants. The enzymes catalyze the hydrolytic removal of ␤ -D -glucosyl residues from nonreducing termini of a range of ␤ -D -glucans and ␤ -D -oligoglucosides. Their broad specificity can be explained by x-ray crystallographic data obtained from a barley ␤ -D -glucan glucohydrolase in complex with nonhydrolyzable S -glycoside substrate analogs and by molecular modeling of enzyme/substrate complexes. The glucosyl residue that occupies binding subsite ؊ 1 is locked tightly into a fixed position through extensive hydrogen bonding with six amino acid residues near the bottom of an active site pocket. In contrast, the glucosyl residue at subsite ؉ 1 is located between two Trp residues at the entrance of the pocket, where it is constrained less tightly. The relative flexibility of binding at subsite ؉ 1, coupled with the projection of the remainder of bound substrate away from the enzyme's surface, means that the overall active site can accommodate a range of substrates with variable spatial dispositions of adjacent ␤ -D -glucosyl residues. The broad specificity for glycosidic linkage type enables the enzyme to perform diverse functions during plant development. INTRODUCTION␤ -D -Glucan glucohydrolases have been purified and characterized from barley seedlings, maize coleoptiles, soybean cultures, Acacia cells, nasturtium cells, and cultured tobacco cells (Cline and Albersheim, 1981;Nari et al., 1982;Lienart et al., 1986;Labrador and Nevins, 1989;Hrmova et al., 1996;Kotake et al., 1997;Crombie et al., 1998;Kim et al., 2000;Koizumi et al., 2000). They can hydrolyze glycosidic linkages in several ␤ -D -glucans, in ␤ -D -oligoglucosides containing (1 → 2)-, (1 → 3)-, (1 → 4)-, or (1 → 6)-linkages, in aryl ␤ -D -glucosides such as 4 Ј -nitrophenyl ␤ -D -glucopyranoside (4NPGlc), and in some ␤ -D -oligoxyloglucosides (Crombie et al., 1998;Hrmova and Fincher, 1998;Kim et al., 2000). The barley ␤ -D -glucan glucohydrolases also hydrolyze cyanogenic ␤ -D -glucosides, albeit with low activity (M. Hrmova and G.B. Fincher, unpublished data). Single Glc molecules are released from the nonreducing termini of these substrates, with retention of the anomeric configuration (Hrmova et al., 1996). Their broad substrate specificity makes it difficult to assign these higher plant ␤ -D -glucan glucohydrolases to current Enzyme Commission classes; therefore, they have been described variously as ␤ -D -glucan glucohydrolases, (1 → 3)-␤ -D -glucan exohydrolases, and ␤ -D -glucosidases. Nevertheless, they can be classified according to the structural criteria of Henrissat (1991) and fall into the family 3 group of glycoside hydrolases (http://afmb.cnrsmrs.fr/CAZY/).The distribution of the broad-specificity ␤ -D -glucan glucohydrolases in various tissues of monocotyledons and dicotyledons, together with the presence of expressed sequence tags in gymnosperm sequence databases (e.g., Pinus taeda ), suggests that they may play a fundamental role in plant growth and development...

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Section: Synthesis Of 4-np S-(␤-d-glucopyranosyl)-(1→3)-(3-thio-␤-d-gmentioning

confidence: 89%

Section: Preparation Of 4-np S-(␤-d-glucopyranosyl)-(1→3)-(3-thio-mentioning

confidence: 99%

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confidence: 99%

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Structural Basis for Broad Substrate Specificity in Higher Plant β-d-Glucan Glucohydrolases

et al. 2002

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“…20) Subsequently, several glycosynthases derived from variousglycosidases have been reported from both exo- [21][22][23][24][25][26] and endo-hydrolases. [27][28][29][30][31][32][33] An -glycoside-forming glycosynthase was also demonstrated with an -glucosidase belonging to GH 31. 34) Recently, we reported the first glycosynthase derived from an inverting enzyme, reducing-end-xylose releasing exo-oligoxylanase, belonging to GH 8.…”

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confidence: 99%

Characterization of Glycosynthase Mutants Derived from Glycoside Hydrolase Family 10 Xylanases

Sugimura

Nishimoto

Kitaoka

2006

Bioscience, Biotechnology, and Biochemistry

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“…When using activated glycosyl donors of the opposite anomeric configuration (compared with the normal substrates) together with suitable acceptors, these mutant enzymes can efficiently synthesize oligosaccharides but without hydrolyzing the resulting products. Since first demonstrated by Withers and co-workers on ␤-glycosidase (11), this technique was applied successfully to a number of other glycoside hydrolases (22)(23)(24). The ␣-L-arabinofuranosidases display several unique features making them attractive candidates for glycosynthesis.…”

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Detailed Kinetic Analysis and Identification of the Nucleophile in α-l-Arabinofuranosidase from Geobacillus stearothermophilus T-6, a Family 51 Glycoside Hydrolase

Shallom¹,

Belakhov²,

Solomon³

et al. 2002

Journal of Biological Chemistry

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␣-L-Arabinofuranosidases cleave the L-arabinofuranoside side chains of different hemicelluloses and are key enzymes in the complete degradation of the plant cell wall. The ␣-L-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase, was subjected to a detailed mechanistic study. Aryl-␣-Larabinofuranosides with various leaving groups were synthesized and used to verify the catalytic mechanism and catalytic residues of the enzyme. The steady-state constants and the resulting Brønsted plots for the E175A mutant are consistent with the role of Glu-175 as the acid-base catalytic residue. The proposed nucleophile residue, Glu-294, was replaced to Ala by a double-base pairs substitution. The resulting E294A mutant, with 4-nitrophenyl ␣-L-arabinofuranoside as the substrate, exhibited eight orders of magnitude lower activity and a 10-fold higher K m value compared with the wild type enzyme. Sodium azide accelerated by more than 40-fold the rate of the hydrolysis of 2 ,4 ,6 -trichlorophenyl ␣-Larabinofuranoside by the E294A mutant. The glycosylazide product formed during this reaction was isolated and characterized as ␤-L-arabinofuranosyl-azide by 1 H NMR, 13 C NMR, mass spectrometry, and Fourier transform infrared analysis. The anomeric configuration of this product supports the assignment of Glu-294 as the catalytic nucleophile residue of the ␣-L-arabinofuranosidase T-6 and allows for the first time the unequivocal identification of this residue in glycoside hydrolases family 51.␣-L-Arabinofuranosidases (EC 3.2.1.55) are hemicellulases that cleave the glycosidic bond between L-arabinofuranosides side chains and different oligosaccharides. These enzymes are part of an array of glycoside hydrolases responsible for the degradation of hemicelluloses such as arabinoxylan, arabinogalactan, and L-arabinan (1-3). The L-arabinofuranoside substitutions on xylans can strongly inhibit the action of endoxylanases and ␤-xylosidases, thus preventing the complete degradation of the polymer to its basic xylose units (4, 5). In many cases, microorganisms that utilize hemicelluloses possess ␣-L-arabinofuranosidases with various substrate specificities and biochemical properties (6). To date, there are more than 110 sequences of different ␣-L-arabinofuranosidases, which are classified, based on sequence homology, into four glycoside hydrolase families (GHs) 1 : GH43, GH51, GH54, and GH62 (7,8). Hemicellulases, together with cellulases, have a key role in the carbon cycle in nature, because they are responsible for the complete degradation of the plant biomass to soluble saccharides. These in turn can be used as carbon or energy sources for microorganisms and higher animals. Hemicellulases have attracted much attention in recent years because of their potential industrial use in biobleaching of paper pulp, bioconversion of lignocellulose material to fermentative products, improvement of animal feedstock digestibility, and organic synthesis (6, 9 -11).The glycosidic bond is one of the most stable bonds in...

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Highly Efficient Synthesis of β(1 → 4)-Oligo- and -Polysaccharides Using a Mutant Cellulase

Cited by 142 publications

References 40 publications

Structural Basis for Broad Substrate Specificity in Higher Plant β-d-Glucan Glucohydrolases

Structural Basis for Broad Substrate Specificity in Higher Plant β-d-Glucan Glucohydrolases

Characterization of Glycosynthase Mutants Derived from Glycoside Hydrolase Family 10 Xylanases

Detailed Kinetic Analysis and Identification of the Nucleophile in α-l-Arabinofuranosidase from Geobacillus stearothermophilus T-6, a Family 51 Glycoside Hydrolase

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