Computer-aided engineering of a branching sucrase for the glucodiversification of a tetrasaccharide precursor of S. flexneri antigenic oligosaccharides

Abstract

Enzyme engineering approaches have allowed to extend the collection of enzymatic tools available for synthetic purposes. However, controlling the regioselectivity of the reaction remains challenging, in particular when dealing with carbohydrates bearing numerous reactive hydroxyl groups as substrates. Here, we used a computer-aided design framework to engineer the active site of a sucrose-active $$\mathrm{\alpha }$$ α -transglucosylase for the 1,2-cis-glucosylation of a lightly protected chemically synthesized tetrasaccharide, a common precursor for the synthesis of serotype-specific S. flexneri O-antigen fragments. By targeting 27 amino acid positions of the acceptor binding subsites of a GH70 branching sucrase, we used a RosettaDesign-based approach to propose 49 mutants containing up to 15 mutations scattered over the active site. Upon experimental evaluation, these mutants were found to produce up to six distinct pentasaccharides, whereas only two were synthesized by the parental enzyme. Interestingly, we showed that by introducing specific mutations in the active site of a same enzyme scaffold, it is possible to control the regiospecificity of the 1,2-cis glucosylation of the tetrasaccharide acceptor and produce a unique diversity of pentasaccharide bricks. This work offers novel opportunities for the development of highly convergent chemo-enzymatic routes toward S. flexneri 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

“…Using an enzyme design protocol based on the sampling of mutations on 27 positions and a Rosetta design-based approach, 12 sequences were selected depending on the SF serotype-specific pentasaccharide products: M6 and M14 (SF type I); M18, M21, M23, M28, M30, M31, and M34 (SF2a serotype); M35, M40, and M41 (SF3a serotype). 34 To characterize the exact activity of these enzymes, a combination of RP−HPLC−UV, RP−HPLC−high-resolution mass spectrometry and NMR was used. This complex approach led to the identification of multiple glucosylation products per enzymes.…”

“…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.…”

“…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.…”

Artificial Neural Network to Predict Structure-based Protein-protein Free Energy of Binding from Rosetta-calculated Properties

“…[1][2][3][4][5] To characterize this association, Gibbs free energy (∆G) of binding is one of the most fundamental thermodynamic quantities. In addition to the comprehension of biomolecular processes, the development of novel biomaterials (e.g., towards biomedical, 6 vaccinal, 7,8 catalytic, 9 development of biosensors, 10 and industrial applications 11 ) relies substantially on the binding affinity (k D ), which is directly related to the ∆G between the involved binding partners (∆G=−RT lnk D , where T is the absolute temperature and R is the universal gas constant).…”