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

• Published in: Chemistry : a European journal Vol. 27; no. 40; pp. 10323 - 10334
• Main Authors: Teze, David, Zhao, Jiao, Wiemann, Mathias, Kazi, Zubaida G. A., Lupo, Rossana, Zeuner, Birgitte, Vuillemin, Marlène, Rønne, Mette E., Carlström, Göran, Duus, Jens Ø., Sanejouand, Yves‐Henri, O'Donohue, Michael J., Nordberg Karlsson, Eva, Fauré, Régis, Stålbrand, Henrik, Svensson, Birte
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 16-07-2021; Wiley-VCH Verlag
• Subjects: Biochemistry; Biochemistry and Molecular Biology; Biochemistry, Molecular Biology; Biokemi och molekylärbiologi; Biologi; Biological Sciences; Biotechnology; Catalysis; Catalysts; Chemical Sciences; Chemistry; enzyme catalysis; Enzymes; Glycosidases; Glycoside hydrolase; Glycosylation; hydrolases; Life Sciences; multiple sequences alignment; Mutation; Natural Sciences; Naturvetenskap; oligosaccharide synthesis; Oligosaccharides; Protein engineering; Proteins; transglycosylation; glycoside hydrolase; protein engineering; transglycosylation; multiple sequences alignment; oligosaccharide synthesis; Transglycosylation; Oligosaccharide synthesis; Protein engineering; Glycoside hydrolases; Enzyme catalysis; Multiple sequence alignment
• ISSN: 0947-6539; 1521-3765; 1521-3765
• DOI: 10.1002/chem.202100110

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.
Although glycosylation is common in Nature, it remains a challenge for organic chemistry. Herein, a method to obtain transglycosylases from retaining glycoside hydrolases is described. The approach pinpoints 6–12 single‐point mutants to assay. Invariably across six GH families, this generates enzyme variants that display increased transglycosylation yields, an improvement that is mostly due to a relative decrease in hydrolysis compared to transglycosylation. The method only requires sequence information, is fast and applicable to a broad range of glycosides.