Design of α-Transgiucosidases of Controlled Specificity for Programmed Chemoenzymatic Synthesis of Antigenic Oligosaccharides

Combined with chemical synthesis, the use of biocatalysts holds great potential to open the way to novel molecular diversity. We report in vitro chemoenzymatic pathways that, for the first time, take advantage of enzyme engineering to produce complex microbial cell-surface oligosaccharides and circumvent the chemical boundaries of glycochemistry. Glycoenzymes were designed to act on nonnatural conveniently protected substrates to produce intermediates compatible with a programmed chemical elongation. The study was focused on the synthesis of oligosaccharides mimicking the O-antigen motif of Shigella flexneri serotypes 1b and 3a, which could be used for the development of multivalent carbohydrate-based vaccines. A semirational engineering approach was successfully applied to amylosucrase, a transglucosidase that uses a low cost sucrose substrate as a glucosyl donor. The main difficulty was to retain the enzyme specificity toward sucrose, while creating a new catalytic function to render the enzyme able to regiospecifically glucosylate protected nonnatural acceptors. A structurally guided library of 133 mutants was generated from which several mutants with either completely new specificity toward methyl alpha-L-rhamnopyranoside or a tremendously enhanced one toward allyl 2-acetamido-2-deoxy-alpha-D-glucopyranoside acceptors were isolated. The best variants were used to synthesize glucosylated building blocks. They were then converted into acceptors and potential donors compatible with chemical elongation toward oligosaccharide fragments of the O-antigens of the two targeted serotypes. This is the first report of a successful engineering of an alpha-transglycosidase acceptor binding site that led to new specificities. It demonstrates the potential of appropriate combinations of a planned chernoenzymatic pathway and enzyme engineering in glycochemistry.