Redirecting substrate regioselectivity using engineered ΔN 123 -GBD-CD2 branching sucrases for the production of pentasaccharide repeating units of S. flexneri 3a, 4a and 4b haptens.

Autor: Benkoulouche M; Toulouse Biotechnology Institute, TBI, Université de Toulouse, CNRS, INRAE, INSA, 135, avenue de Rangueil, 31077, Toulouse Cedex 04, France., Ben Imeddourene A; Toulouse Biotechnology Institute, TBI, Université de Toulouse, CNRS, INRAE, INSA, 135, avenue de Rangueil, 31077, Toulouse Cedex 04, France., Barel LA; Unité de Chimie des Biomolécules, Institut Pasteur, UMR3523 CNRS, 28, rue du Dr Roux, 75724, Paris Cedex 15, France.; Université Paris Descartes, Sorbonne Paris Cité, Paris, France., Le Heiget G; Unité de Chimie des Biomolécules, Institut Pasteur, UMR3523 CNRS, 28, rue du Dr Roux, 75724, Paris Cedex 15, France.; Université Paris Descartes, Sorbonne Paris Cité, Paris, France.; Université Paris 13, Bobigny, France., Pizzut S; Toulouse Biotechnology Institute, TBI, Université de Toulouse, CNRS, INRAE, INSA, 135, avenue de Rangueil, 31077, Toulouse Cedex 04, France., Kulyk H; Toulouse Biotechnology Institute, TBI, Université de Toulouse, CNRS, INRAE, INSA, 135, avenue de Rangueil, 31077, Toulouse Cedex 04, France.; MetaboHUB-MetaToul, National Infrastructure for Metabolomics and Fluxomics, Toulouse, France., Bellvert F; Toulouse Biotechnology Institute, TBI, Université de Toulouse, CNRS, INRAE, INSA, 135, avenue de Rangueil, 31077, Toulouse Cedex 04, France.; MetaboHUB-MetaToul, National Infrastructure for Metabolomics and Fluxomics, Toulouse, France., Bozonnet S; Toulouse Biotechnology Institute, TBI, Université de Toulouse, CNRS, INRAE, INSA, 135, avenue de Rangueil, 31077, Toulouse Cedex 04, France., Mulard LA; Unité de Chimie des Biomolécules, Institut Pasteur, UMR3523 CNRS, 28, rue du Dr Roux, 75724, Paris Cedex 15, France., Remaud-Siméon M; Toulouse Biotechnology Institute, TBI, Université de Toulouse, CNRS, INRAE, INSA, 135, avenue de Rangueil, 31077, Toulouse Cedex 04, France., Moulis C; Toulouse Biotechnology Institute, TBI, Université de Toulouse, CNRS, INRAE, INSA, 135, avenue de Rangueil, 31077, Toulouse Cedex 04, France. claire.moulis@insa-toulouse.fr., André I; Toulouse Biotechnology Institute, TBI, Université de Toulouse, CNRS, INRAE, INSA, 135, avenue de Rangueil, 31077, Toulouse Cedex 04, France. isabelle.andre@insa-toulouse.fr.
Jazyk: angličtina
Zdroj: Scientific reports [Sci Rep] 2021 Jan 28; Vol. 11 (1), pp. 2474. Date of Electronic Publication: 2021 Jan 28.
DOI: 10.1038/s41598-021-81719-1
Abstrakt: The (chemo-)enzymatic synthesis of oligosaccharides has been hampered by the lack of appropriate enzymatic tools with requisite regio- and stereo-specificities. Engineering of carbohydrate-active enzymes, in particular targeting the enzyme active site, has notably led to catalysts with altered regioselectivity of the glycosylation reaction thereby enabling to extend the repertoire of enzymes for carbohydrate synthesis. Using a collection of 22 mutants of ΔN 123 -GBD-CD2 branching sucrase, an enzyme from the Glycoside Hydrolase family 70, containing between one and three mutations in the active site, and a lightly protected chemically synthesized tetrasaccharide as an acceptor substrate, we showed that altered glycosylation product specificities could be achieved compared to the parental enzyme. Six mutants were selected for further characterization as they produce higher amounts of two favored pentasaccharides compared to the parental enzyme and/or new products. The produced pentasaccharides were shown to be of high interest as they are precursors of representative haptens of Shigella flexneri serotypes 3a, 4a and 4b. Furthermore, their synthesis was shown to be controlled by the mutations introduced in the active site, driving the glucosylation toward one extremity or the other of the tetrasaccharide acceptor. To identify the molecular determinants involved in the change of ΔN 123 -GBD-CD2 regioselectivity, extensive molecular dynamics simulations were carried out in combination with in-depth analyses of amino acid residue networks. Our findings help to understand the inter-relationships between the enzyme structure, conformational flexibility and activity. They also provide new insight to further engineer this class of enzymes for the synthesis of carbohydrate components of bacterial haptens.
Databáze: MEDLINE
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