Redirecting substrate regioselectivity using engineered \u0394N123-GBD-CD2 branching sucrases for the production of pentasaccharide repeating units of S. flexneri 3a, 4a and 4b haptens

Mounir Benkoulouche,Claire Moulis,Magali Remaud-Siméon,Louis-Antoine Barel,Laurence A Mulard,Sandra Pizzut,Akli Ben Imeddourene,Guillaume Le Heiget,Sophie Bozonnet,Floriant Bellvert,Hanna Kulyk,Isabelle André

doi:10.1038/s41598-021-81719-1

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.

Highlights

Carbohydrate-active enzymes catalyze a wide range of chemical reactions
Our group has been active in this field, notably by engineering sucrose-active α-transglucosylases from Glycoside Hydrolase (GH) families 13 and 70 of the CAZy classification[2], to produce a variety of carbohydrate derivatives and glycoconjugates[3,4,5,6,7]
In the continuity of these studies, we recently tested the ability of branching sucrases—native GH70 sucrose-active transglucosylases specialized in dextran branching—to regioselectively glucosylate a lightly protected tetrasaccharide (allyl α-l-rhamnopyranosyl-(1 → 2)-α-l-rhamnopyranosyl-(1 → 3)-(2-O-chloroacetylα-l-rhamnopyranosyl)-(1 → 3)-2-deoxy-2-trichloroacetamido-β-d-glucopyranoside, named ABC′D′), which was designed and chemically synthesized to serve as a common scaffold to precursors of Shigella flexneri serotype-specific haptens that could enter in the composition of a broad serotype coverage Shigella vaccine, while respecting criteria imposed by the enzyme plasticity

Summary

Introduction

Carbohydrate-active enzymes catalyze a wide range of chemical reactions. They have emerged as a practical alternative to chemical catalysts, avoiding multiple steps of protection and deprotection often required in chemical synthesis to control the reactivity of the sugar hydroxyl groups and regio- and stereo-selectivity of the reaction. Detailed molecular dynamics through multi-level modelling methods revealed the high flexibility of several loops surrounding the catalytic pocket, which could play a beneficial role in the recognition of a broader range of a cceptors[11] To support this assumption, (semi)rational engineering of ΔN123-GBD-CD2 enabled the diversification of bulky flavonoid glucosides[6] by use of a small mutant library targeting mutations four amino acid residues from acceptor subsites + 1 and + 2 of the catalytic pocket (Supplementary Fig. S1). This study highlights the potential of GH70 branching sucrase engineering for glucodiversification of a S. flexneri tetrasaccharide backbone as well as to control the reaction regioselectivity These promising results pave the way for a more exhaustive reshaping of branching sucrase active site to further extend the panel of accessible glucosylation patterns and provide synthetic tools for the development of a broad coverage synthetic glycan-based vaccine against S. flexneri

Methods

Results

Conclusion