Rational Enzyme Design without Structural Knowledge: A Sequence‐Based Approach for Efficient Generation of Transglycosylases

Abstract

Glycobiology is dogged by the relative scarcity of synthetic, defined oligosaccharides. Enzyme-catalysed glycosylation using glycoside hydrolases is feasible but is hampered by the innate hydrolytic activity of these enzymes. Protein engineering is useful to remedy this, but it usually requires prior structural knowledge of the target enzyme, and/or relies on extensive, time-consuming screening and analysis. Here, a straightforward strategy that involves rational rapid in silico analysis of protein sequences is described. The method pinpoints 6-12 single-mutant candidates to improve transglycosylation yields. Requiring very little prior knowledge of the target enzyme other than its sequence, the method is generic and procures catalysts for the formation of glycosidic bonds involving various d/l-, α/β-pyranosides or furanosides, and exo or endo action. Moreover, mutations validated in one enzyme can be transposed to others, even distantly related enzymes.

Improvement of the Transglycosylation Efficiency of a Lacto-N-Biosidase from Bifidobacterium bifidum by Protein Engineering

“…3.1.2. Conserved Residues: W373F, W373H, W394F, W394H, W465F, W465H, and D467N LNT formation was not significantly improved for the D467N variant (Table 1; Figure S4), which is in line with the observations on the equivalent BbhI variant D884N [26]. The total transglycosylation yield did improve compared to the WT, but regioselectivity was low (only half of the formed product was LNT; Table 1).…”

“…The catalytic Asp-Glu pair is highlighted in red, while the substrate-stabilizing Tyr is marked in purple; mutation of the latter improved transglycosylation in BbhI, LnbB, and Tf Hex [15,17,22]. Conserved residues found to improve BbhI transglycosylation yield through conservative substitutions are marked in green, whereas those marked in blue did not improve BbhI transglycosylation yields [26]. An additional, conserved residue (W373) identified in the current work by multiple sequence alignment of all GH20s characterized in the CAZy database is highlighted in grey.…”

Two marine GH29 α-L-fucosidases from an uncultured Paraglaciecola sp. specifically hydrolyze fucosyl-N-acetylglucosamine regioisomers

“…mutagenesis, change of reaction conditions, increasing the substrate concentration and so forth (Bissaro et al 2015;Zeuner et al 2019;Teze et al 2021). In this context, AlfB and AlfC from L. casei by reaction with pNP-α-L-fucose and GlcNAc generated Fuc(α1,3)GlcNAc and Fuc(α1,6)GlcNAc, respectively, which are important building blocks in HMOs or found in glucoconjugates at mucosal surfaces (Rodriguez-Diaz et al 2013;Teze et al 2021). Fuc(α1,4)GlcNAc has similar biological importance, and therefore Fp231 was investigated for ability to catalyze formation of Fuc(α1,4)GlcNAc and Fuc(α1,4)GlcNAc-linked products.…”

“…Unfortunately, no transglycosylation products were observed when using wild type Fp231 with 20 mM CNP-Fuc as donor and 200 mM GlcNAc as acceptor (data not shown). In an attempt to stimulate transglycosylation, Fp231 mutants H174F and W225H were designed by using mutant transfer based on a strategy of conserved-residue mutation, which enabled in this case increased transglycosylation by both L. casei AlfB and AlfC of GH29 (Teze et al 2021). However, still no transglycosylation product was obtained.…”

“…The gene inserts in purified plasmids were verified by Sanger sequencing (Eurofins Genomics, Germany) and transformed into E. coli BL21 (DE3) (New England Biolabs). In an attempt to improve transglycosylation, the mutants Fp231_H174F and Fp231_W225H were designed based on mutant transfer (Teze et al 2021) and introduced into the Fp231 gene as described (Liu and Naismith 2008) using the primers listed in Supplementary Table I.…”