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

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 cata...

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Autores principales: Zeuner, Birgitte, Vuillemin, Marlene, Holck, Jesper, Muschiol, Jan, Meyer, Anne S.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7711723/
https://www.ncbi.nlm.nih.gov/pubmed/33217923
http://dx.doi.org/10.3390/jof6040295
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author Zeuner, Birgitte
Vuillemin, Marlene
Holck, Jesper
Muschiol, Jan
Meyer, Anne S.
author_facet Zeuner, Birgitte
Vuillemin, Marlene
Holck, Jesper
Muschiol, Jan
Meyer, Anne S.
author_sort Zeuner, Birgitte
collection PubMed
description 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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spelling pubmed-77117232020-12-04 Improved Transglycosylation by a Xyloglucan-Active α-l-Fucosidase from Fusarium graminearum Zeuner, Birgitte Vuillemin, Marlene Holck, Jesper Muschiol, Jan Meyer, Anne S. J Fungi (Basel) Article 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. MDPI 2020-11-18 /pmc/articles/PMC7711723/ /pubmed/33217923 http://dx.doi.org/10.3390/jof6040295 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Zeuner, Birgitte
Vuillemin, Marlene
Holck, Jesper
Muschiol, Jan
Meyer, Anne S.
Improved Transglycosylation by a Xyloglucan-Active α-l-Fucosidase from Fusarium graminearum
title Improved Transglycosylation by a Xyloglucan-Active α-l-Fucosidase from Fusarium graminearum
title_full Improved Transglycosylation by a Xyloglucan-Active α-l-Fucosidase from Fusarium graminearum
title_fullStr Improved Transglycosylation by a Xyloglucan-Active α-l-Fucosidase from Fusarium graminearum
title_full_unstemmed Improved Transglycosylation by a Xyloglucan-Active α-l-Fucosidase from Fusarium graminearum
title_short Improved Transglycosylation by a Xyloglucan-Active α-l-Fucosidase from Fusarium graminearum
title_sort improved transglycosylation by a xyloglucan-active α-l-fucosidase from fusarium graminearum
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7711723/
https://www.ncbi.nlm.nih.gov/pubmed/33217923
http://dx.doi.org/10.3390/jof6040295
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