Redirecting substrate regioselectivity using engineered ΔN123-GBD-CD2 branching sucrases for the production of pentasaccharide repeating units of S. flexneri 3a, 4a and 4b haptens

Abstract 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 alte...

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Autores principales:	Mounir Benkoulouche, Akli Ben Imeddourene, Louis-Antoine Barel, Guillaume Le Heiget, Sandra Pizzut, Hanna Kulyk, Floriant Bellvert, Sophie Bozonnet, Laurence A. Mulard, Magali Remaud-Siméon, Claire Moulis, Isabelle André
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/ef2c88e7c7fc466baca624798a644eb8
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spelling	oai:doaj.org-article:ef2c88e7c7fc466baca624798a644eb82021-12-02T13:24:17ZRedirecting substrate regioselectivity using engineered ΔN123-GBD-CD2 branching sucrases for the production of pentasaccharide repeating units of S. flexneri 3a, 4a and 4b haptens10.1038/s41598-021-81719-12045-2322https://doaj.org/article/ef2c88e7c7fc466baca624798a644eb82021-01-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-81719-1https://doaj.org/toc/2045-2322Abstract 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 ΔN123-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 ΔN123-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.Mounir BenkouloucheAkli Ben ImeddoureneLouis-Antoine BarelGuillaume Le HeigetSandra PizzutHanna KulykFloriant BellvertSophie BozonnetLaurence A. MulardMagali Remaud-SiméonClaire MoulisIsabelle AndréNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-14 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Mounir Benkoulouche Akli Ben Imeddourene Louis-Antoine Barel Guillaume Le Heiget Sandra Pizzut Hanna Kulyk Floriant Bellvert Sophie Bozonnet Laurence A. Mulard Magali Remaud-Siméon Claire Moulis Isabelle André Redirecting substrate regioselectivity using engineered ΔN123-GBD-CD2 branching sucrases for the production of pentasaccharide repeating units of S. flexneri 3a, 4a and 4b haptens
description	Abstract 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 ΔN123-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 ΔN123-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.
format	article
author	Mounir Benkoulouche Akli Ben Imeddourene Louis-Antoine Barel Guillaume Le Heiget Sandra Pizzut Hanna Kulyk Floriant Bellvert Sophie Bozonnet Laurence A. Mulard Magali Remaud-Siméon Claire Moulis Isabelle André
author_facet	Mounir Benkoulouche Akli Ben Imeddourene Louis-Antoine Barel Guillaume Le Heiget Sandra Pizzut Hanna Kulyk Floriant Bellvert Sophie Bozonnet Laurence A. Mulard Magali Remaud-Siméon Claire Moulis Isabelle André
author_sort	Mounir Benkoulouche
title	Redirecting substrate regioselectivity using engineered ΔN123-GBD-CD2 branching sucrases for the production of pentasaccharide repeating units of S. flexneri 3a, 4a and 4b haptens
title_short	Redirecting substrate regioselectivity using engineered ΔN123-GBD-CD2 branching sucrases for the production of pentasaccharide repeating units of S. flexneri 3a, 4a and 4b haptens
title_full	Redirecting substrate regioselectivity using engineered ΔN123-GBD-CD2 branching sucrases for the production of pentasaccharide repeating units of S. flexneri 3a, 4a and 4b haptens
title_fullStr	Redirecting substrate regioselectivity using engineered ΔN123-GBD-CD2 branching sucrases for the production of pentasaccharide repeating units of S. flexneri 3a, 4a and 4b haptens
title_full_unstemmed	Redirecting substrate regioselectivity using engineered ΔN123-GBD-CD2 branching sucrases for the production of pentasaccharide repeating units of S. flexneri 3a, 4a and 4b haptens
title_sort	redirecting substrate regioselectivity using engineered δn123-gbd-cd2 branching sucrases for the production of pentasaccharide repeating units of s. flexneri 3a, 4a and 4b haptens
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/ef2c88e7c7fc466baca624798a644eb8
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Redirecting substrate regioselectivity using engineered ΔN123-GBD-CD2 branching sucrases for the production of pentasaccharide repeating units of S. flexneri 3a, 4a and 4b haptens

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