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 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.

Combination of High-Resolution Multistage Ion Mobility and Tandem MS with High Energy of Activation to Resolve the Structure of Complex Chemoenzymatically Synthesized Glycans

“…Interestingly, the later pattern defines SFtype factor V. Unfortunately discriminating between these two isomers was impossible due to the lack of informative fragments. 34 In order to refine our understanding of the products of ABC′D′ enzymatic α-D-glucosylation, the 14 reaction media issued from the independent reaction of ABC′D′ with the 12 mutants designed from the GH70 branching sucrase BRS-B 34 and 2 mutants from the GH70 branching sucrase ΔN 123 -GBD-CD2, F2163G named G and W2315S-F2136L named SL, 35 were studied using advanced MS-based approaches.…”

“…Crude reaction mixtures of pentasaccharides were obtained from the glucansucrase mutant-mediated α- d -glucosylation of the chemically synthetized tetrasaccharide ABC′D′ (Figure ) as described previously . Biocatalysts used for generating the samples studied in this work were described previously. , …”

“…A collection of 14 computer-aided engineered mutants of branching sucrases from the glycoside hydrolase family 70 (GH70) were selected for their ability to produce specific building blocks involved in the synthesis of haptens representative of the O-antigen of prevalent serotypes of Shigella flexneri (SF). 34,35 one for which there is no licensed vaccine. 36,37 Reaction media were studied using a unique multipass high-resolution IMS setup and CTD activation, both combined with a PGC− UHPLC strategy.…”

“…38 Biocatalysts used for generating the samples studied in this work were described previously. 34,35 Chemicals. HPLC grade acetonitrile (ACN) was purchased from Carlo-Erba.…”

“…In the present work, complex pentasaccharides obtained from the enzymatic α- d -glucosylation of a non-natural lightly protected tetrasaccharide acceptor were structurally characterized. A collection of 14 computer-aided engineered mutants of branching sucrases from the glycoside hydrolase family 70 (GH70) were selected for their ability to produce specific building blocks involved in the synthesis of haptens representative of the O-antigen of prevalent serotypes of Shigella flexneri (SF). , This family of bacteria is responsible for shigellosis, one of the most critical enteric diseases in children under five in low and middle income countries, and one for which there is no licensed vaccine. , Reaction media were studied using a unique multipass high-resolution IMS setup and CTD activation, both combined with a PGC–UHPLC strategy. Our results show that the emergent methods proposed in this workflow are powerful technologies that can be used to screen complex mixtures and to identify the fine structure of specific glycans with a very high level of certainty, as required in biopharma.…”