Computer-Aided Engineering of a Transglycosylase for the Glucosylation of an Unnatural Disaccharide of Relevance for Bacterial Antigen Synthesis

Abstract

The exploration of chemo-enzymatic routes to complex carbohydrates has been hampered by the lack of appropriate enzymatic tools having the substrate specificity for new reactions. Here, we used a computer-aided design framework to guide the construction of a small, diversity-controlled library of amino acid sequences of an α-transglucosylase, the sugar binding subsites of which were re-engineered to enable the challenging 1,2-cis-glucosylation of a partially protected β-linked disaccharide allyl (2-deoxy-2-trichloroacetamido-β-D-glucopyranosyl)-(1→2)-α-L-rhamnopyranoside, a potential intermediate in the synthesis of Shigella flexneri cell-surface oligosaccharides. The target disaccharide is not recognized by the parental wild-type enzyme and exhibits a molecular structure very distinct from that of the natural α-(1→4)-linked acceptor. A profound reshaping of the binding pocket had thus to be performed. Following the selection of 23 amino acid positions from the first shell, mutations were sampled using RosettaDesign leading to a subset of 1515 designed sequences, which were further analyzed by determining the amino acid variability among the designed sequences and their conservation in evolutionary-related enzymes. A combinatorial library of 2.7 × 10 4 variants was finally designed, constructed, and screened. One mutant showing the desired and totally new specificity was successfully identified from this first round of screening. Impressively, this mutant contained seven substitutions in the first shell of the active site leading to a drastic reshaping of the catalytic pocket without significantly perturbing the original specificity for sucrose donor substrate. This work illustrates how computer-aided approaches can undoubtedly offer novel opportunities to design tailored carbohydrate-active enzymes of interest for glycochemistry or synthetic glycobiology.

Novel product specificity toward erlose and panose exhibited by multisite engineered mutants of amylosucrase

“…Semi‐rational design is widely viewed as a very efficient approach for tailoring substrate or product specificity of enzymes. Applied to Np AS enzyme, a computer aided‐approach to engineer subsites +1 and +2 allowed to isolate one mutant still able to produce soluble maltooligosaccharides, although shorter than for the wild‐type Np AS, from sole sucrose and which also gained a totally new ability to recognize an unnatural and chemically protected disaccharide of interest for novel vaccine elaboration . The redesign of the catalytic pocket led to the isolation of 55 sucrose‐active clones out of 63,000 screened clones.…”

“…The construction of the libraries from which were isolated the mutants characterized herein were previously described . Briefly, the libraries of Np AS were initially designed to recognize and glucosylate a partially protected β‐linked disaccharide acceptor called allyl 2‐deoxy‐2‐ N ‐trichloroacetyl‐β‐D‐glucopyranosyl‐(1→2)‐α‐ l ‐rhamnopyranoside.…”

“…Briefly, the libraries of Np AS were initially designed to recognize and glucosylate a partially protected β‐linked disaccharide acceptor called allyl 2‐deoxy‐2‐ N ‐trichloroacetyl‐β‐D‐glucopyranosyl‐(1→2)‐α‐ l ‐rhamnopyranoside. Altogether, 63,000 variants were screened using a pH‐based assay on solid medium as described earlier . The 55 active clones were stored in 96‐well microplates containing LB medium (200 µL), ampicillin (100 µg mL −1 ) and glycerol (12% w/v).…”

“…Very recently, Np AS active site was deeply reshaped to adapt the enzyme to the glucosylation of unnatural and slightly protected disaccharides entering in a programmed chemo‐enzymatic pathway designed for bacterial antigen synthesis . Using a computer‐aided approach, semi‐rational libraries, totaling about 2.7 × 10 4 variants and targeting 23 amino acid positions of subsites +1 and +2 in the active site first shell, were designed.…”

“…8,10,13,14 Very recently, NpAS active site was deeply reshaped to adapt the enzyme to the glucosylation of unnatural and slightly protected disaccharides entering in a programmed chemo-enzymatic pathway designed for bacterial antigen synthesis. 15 Using a computer-aided approach, semi-rational libraries, totaling about 2.7 3 10 4 variants and targeting 23 amino acid positions of subsites 11 and 12 in the active site first shell, were designed. Upon library construction, the mutants were screened, first, on sucrose activity and, next, on their ability to catalyze the glucosylation of the target acceptor, a partially protected b-linked disaccharide, the allyl (2-deoxy-2-trichloroacetamido-b-D-glucopyranosyl)-(1!2)-a-L-rhamnopyranoside.…”