Taxonomic group: bacteria / Proteobacteria
(Phylum: Proteobacteria)
Associated disease: infection due to Shigella flexneri [ICD11:
XN7Y2 
]
NCBI PubMed ID: 34645865Publication DOI: 10.1038/s41598-021-99384-9Journal NLM ID: 101563288Publisher: London: Nature Publishing Group
Correspondence: isabelle.andre
insa-toulouse.fr
Institutions: Toulouse Biotechnology Institute, TBI, Université de Toulouse, CNRS, INRAE, INSA, 135, Avenue de Rangueil, 31077, Toulouse Cedex 04, France, Institut Pasteur, CNRS UMR3523 Unité de Chimie des Biomolécules, 28 Rue du Dr Roux, 75724, Paris Cedex 15, France, Université Paris Descartes, Sorbonne Paris Cité, Paris, France, INRAE, UR BIA, 44316, Nantes, France, INRAE, BIBS Facility, 44316, Nantes, France
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 [Formula: see text]-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.
engineering, Haptens, computer-aided design, S.flexneri
Structure type: polymer chemical repeating unit
Location inside paper: Fig. 1A
Compound class: O-polysaccharide, O-antigen
Contained glycoepitopes: IEDB_125613,IEDB_125614,IEDB_127514,IEDB_130421,IEDB_130422,IEDB_133752,IEDB_133753,IEDB_133754,IEDB_135813,IEDB_136105,IEDB_137340,IEDB_141807,IEDB_141815,IEDB_141816,IEDB_142488,IEDB_143253,IEDB_144998,IEDB_146664,IEDB_151531,IEDB_153213,IEDB_158539,IEDB_225177,IEDB_885823,IEDB_983931,SB_192
Methods: 13C NMR, 1H NMR, PCR, chemical synthesis, genetic methods, computational methods, HPLC-UV-MS, UHPLC-MS/MS
3D data: 3D model of BRS-B Δ2 enzyme (branching sucrase)
NCBI Taxonomy refs (TaxIDs): 424717Reference(s) to other database(s): GTC:G69624OZ, GlycomeDB:
37120
Show glycosyltransferases
There is only one chemically distinct structure:
Found 
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