Engineering faster transglycosidases and their acceptor specificity

Tran, Linh Thuy Thi; Blay, Vincent; Luang, Sukanya; Eurtivong, Chatchakorn; Choknud, Sunaree; González-Dı́az, Humberto; Cairns, James R. Ketudat

doi:10.1039/c9gc00621d

Cited by 15 publications

(12 citation statements)

References 53 publications

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“…Directed evolution by random mutagenesis can be a powerful tool for identifying mutations, which improve transglycosylation significantly [ 13 , 24 , 33 ]. However, the fact that neither Fg FCO1 nor any GH29B members are active on the chromogenic substrate p NP-α- l -Fuc [ 7 ] complicates the screening process, since chromogenic substrates are most often used in the first mutant screening [ 13 , 22 , 23 , 24 ]. Instead, we aimed to transfer successful engineering results obtained on other GH29 α- l -fucosidases—including the GH29A Tm αFuc from Thermotoga maritima , which is active on p NP-α- l -Fuc—to the xyloglucan-active Fg FCO1 by rational design.…”

Section: Resultsmentioning

confidence: 99%

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Improved Transglycosylation by a Xyloglucan-Active α-l-Fucosidase from Fusarium graminearum

Zeuner

Vuillemin

Holck

et al. 2020

JoF

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Fusarium graminearum produces an α-l-fucosidase, FgFCO1, which so far appears to be the only known fungal GH29 α-l-fucosidase that catalyzes the release of fucose from fucosylated xyloglucan. In our quest to synthesize bioactive glycans by enzymatic catalysis, we observed that FgFCO1 is able to catalyze a transglycosylation reaction involving transfer of fucose from citrus peel xyloglucan to lactose to produce 2′-fucosyllactose, an important human milk oligosaccharide. In addition to achieving maximal yields, control of the regioselectivity is an important issue in exploiting such a transglycosylation ability successfully for glycan synthesis. In the present study, we aimed to improve the transglycosylation efficiency of FgFCO1 through protein engineering by transferring successful mutations from other GH29 α-l-fucosidases. We investigated several such mutation transfers by structural alignment, and report that transfer of the mutation F34I from BiAfcB originating from Bifidobacterium longum subsp. infantis to Y32I in FgFCO1 and mutation of D286, near the catalytic acid/base residue in FgFCO1, especially a D286M mutation, have a positive effect on FgFCO1 transfucosylation regioselectivity. We also found that enzymatic depolymerization of the xyloglucan substrate increases substrate accessibility and in turn transglycosylation (i.e., transfucosylation) efficiency. The data include analysis of the active site amino acids and the active site topology of FgFCO1 and show that transfer of point mutations across GH29 subfamilies is a rational strategy for targeted protein engineering of a xyloglucan-active fungal α-l-fucosidase.

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Section: Resultsmentioning

confidence: 99%

“…Setting up a high-throughput screening system for directed evolution is complicated for transglycosylation, which takes place in competition with hydrolysis, but can be achieved if the GH is active on a chromogenic substrate [ 13 , 22 , 23 , 24 ]. However, Fg FCO1 is not active on p NP-α- l -Fuc [ 7 , 25 ].…”

Section: Introductionmentioning

confidence: 99%

Improved Transglycosylation by a Xyloglucan-Active α-l-Fucosidase from Fusarium graminearum

Zeuner

Vuillemin

Holck

et al. 2020

JoF

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“…Transglycosidases (TGs) catalyze the transfer of a glycosyl unit from a donor glycoside (often readily available and cheap, unlike nucleotide sugars) onto an acceptor molecule, to produce another type of carbohydrate or glycoconjugate, thanks to their wide acceptor promiscuity. More and more attention is being given to the discovery of new TGs [ 11 ] and their engineering [ 12 , 13 , 14 , 15 ], for oligosaccharide synthesis in vitro. However, only a few native TGs have been described, and their specificities are restricted to some substrates.…”

Section: Introductionmentioning

confidence: 99%

Discovery and Biotechnological Exploitation of Glycoside-Phosphorylases

Benkoulouche

Ladevèze

et al. 2022

IJMS

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Among carbohydrate active enzymes, glycoside phosphorylases (GPs) are valuable catalysts for white biotechnologies, due to their exquisite capacity to efficiently re-modulate oligo- and poly-saccharides, without the need for costly activated sugars as substrates. The reversibility of the phosphorolysis reaction, indeed, makes them attractive tools for glycodiversification. However, discovery of new GP functions is hindered by the difficulty in identifying them in sequence databases, and, rather, relies on extensive and tedious biochemical characterization studies. Nevertheless, recent advances in automated tools have led to major improvements in GP mining, activity predictions, and functional screening. Implementation of GPs into innovative in vitro and in cellulo bioproduction strategies has also made substantial advances. Herein, we propose to discuss the latest developments in the strategies employed to efficiently discover GPs and make the best use of their exceptional catalytic properties for glycoside bioproduction.

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“…The factors considered in the selection of amino acids for this purpose include the location of flexible regions associated with internal water transport [46,47], shifts in acid-baseresidue dynamics [47], and sidechain conformational changes of residues near the active center [48]. Some other investigations have intended to modify the specificity of the acceptor site toward diverse organic molecules [49] and have succeeded in changing the T/H ratio; nevertheless, the strategies explored have generally focused on punctual mutations near the active site identified by multiple sequence alignments (MSA) of a few glycosidasespresumably with high specificity towards hydrolytic or transfer reactions [43,45,[50][51][52].…”

Section: Introductionmentioning

confidence: 99%

Modulating Glycoside Hydrolase Activity between Hydrolysis and Transfer Reactions Using an Evolutionary Approach

et al. 2021

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The proteins within the CAZy glycoside hydrolase family GH13 catalyze the hydrolysis of polysaccharides such as glycogen and starch. Many of these enzymes also perform transglycosylation in various degrees, ranging from secondary to predominant reactions. Identifying structural determinants associated with GH13 family reaction specificity is key to modifying and designing enzymes with increased specificity towards individual reactions for further applications in industrial, chemical, or biomedical fields. This work proposes a computational approach for decoding the determinant structural composition defining the reaction specificity. This method is based on the conservation of coevolving residues in spatial contacts associated with reaction specificity. To evaluate the algorithm, mutants of α-amylase (TmAmyA) and glucanotransferase (TmGTase) from Thermotoga maritima were constructed to modify the reaction specificity. The K98P/D99A/H222Q variant from TmAmyA doubled the transglycosydation/hydrolysis (T/H) ratio while the M279N variant from TmGTase increased the hydrolysis/transglycosidation ratio five-fold. Molecular dynamic simulations of the variants indicated changes in flexibility that can account for the modified T/H ratio. An essential contribution of the presented computational approach is its capacity to identify residues outside of the active center that affect the reaction specificity.

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Engineering faster transglycosidases and their acceptor specificity

Abstract: Transglycosidases have potential to catalyze the synthesis of high-value compounds from biomass-derived feedstocks. Cheminformatics can help design more active and versatile catalysts and discover new substrates.

Cited by 15 publications

References 53 publications

Improved Transglycosylation by a Xyloglucan-Active α-l-Fucosidase from Fusarium graminearum

Improved Transglycosylation by a Xyloglucan-Active α-l-Fucosidase from Fusarium graminearum

Discovery and Biotechnological Exploitation of Glycoside-Phosphorylases

Modulating Glycoside Hydrolase Activity between Hydrolysis and Transfer Reactions Using an Evolutionary Approach

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