Engineering a branching sucrase for flavonoid glucoside diversification

Abstract

Enzymatic glycosylation of flavonoids is an efficient mean to protect aglycons against degradation while enhancing their solubility, life time and, by extension, their bioavailability which is critical for most of their applications in health care. To generate a valuable enzymatic platform for flavonoid glucosylation, an α-1,2 branching sucrase belonging to the family 70 of glycoside-hydrolases was selected as template and subsequently engineered. Two libraries of variants targeting pair-wise mutations inferred by molecular docking simulations were generated and screened for quercetin glucosylation using sucrose as a glucosyl donor. Only a limited number of variants (22) were retained on the basis of quercetin conversion and product profile. Their acceptor promiscuity towards five other flavonoids was subsequently assessed, and the automated screening effort revealed variants showing remarkable ability for luteolin, morin and naringenin glucosylation with conversion ranging from 30% to 90%. Notably, naringenin and morin, a priori considered as recalcitrant compounds to glucosylation using this α-transglucosylases, could also be modified. The approach reveals the potential of small platforms of engineered GH70 α-transglucosylases and opens up the diversity of flavonoid glucosides to molecular structures inaccessible yet.

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

“…Preexistence of such conformation changes is also underlined by the correlation found between the network derived from MD simulations and the product profiles of mutants W2135S-F2136L and W2135I-F2136C obtained from distinct acceptors ( ABC′D′ tetrasaccharide studied herein, Fig. 1 , and flavonoids 6 ).…”

“…S1 ). In the seminal work that led to the construction of the mutant library, they were targeted with the aim of gaining space to better accommodate bulky flavonoid molecules in the acceptor subsites 6 . We hypothesized that the same mutations could also help to accommodate the large tetrasaccharide ABC′D′, which harbors bulky protecting groups at positions 1 D′ , 2 D′ and 2 C′ , in a catalytically productive manner.…”

“…Overall, our study shows how amino acid substitutions introduced in the acceptor subsites + 1, + 2 and + 3 of ΔN 123 -GBD-CD2 branching sucrase could drastically shift the preferred binding mode of a non-natural tetrasaccharide acceptor, a lightly protected precursor in the synthesis of S. flexneri haptens, and thereby control its regioselective glucosylation. From the screening of a focused library of 22 mutants earlier reported 6 , we identified 6 mutants exhibiting singular product profiles with respect to the parental enzyme which was earlier reported to produce in particular a pentasaccharide P1 and an uncharacterized monoglucosylated product P2 . By introducing a limited number of mutations—up to three—it was possible to drive the transglucosylation reaction toward one extremity or the other of the tetrasaccharide ABC′D′ ( A vs D′ ), producing four distinct pentasaccharides ( P1, P2 , P2′ , and P3 ) in different amounts.…”

“…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 acceptors 11 . To support this assumption, (semi)rational engineering of ΔN 123 -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 ).…”

“…active site has been very efficient to alter substrate specificity and expand the structural diversity of the accessible glyco-products. In recent years, our group has been particularly 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 particular, the amylosucrase from Neisseria polysaccharea, a GH13 sucrose-active enzyme, was purposely tailored to act on non-natural substrates and produce glucosylated derivatives programmed to enter various chemo-enzymatic pathways combining in the most convenient manner chemical and enzymatic steps involved in the synthesis of antigenic oligosaccharides.…”