Rational Enzyme Design without Structural Knowledge: A Sequence-Based Approach for Efficient Generation of Transglycosylases.
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.
['Teze|David|D|http://orcid.org/0000-0002-6865-6108', 'Zhao|Jiao|J|http://orcid.org/0000-0002-9731-4473', 'Wiemann|Mathias|M|http://orcid.org/0000-0003-0803-0370', 'Kazi|Zubaida G A|ZGA|http://orcid.org/0000-0003-0929-8045', 'Lupo|Rossana|R|http://orcid.org/0000-0002-0377-3530', 'Zeuner|Birgitte|B|http://orcid.org/0000-0002-8593-6742', 'Vuillemin|Marlène|M|http://orcid.org/0000-0002-1776-7844', 'Rønne|Mette E|ME|http://orcid.org/0000-0002-7892-4655', 'Carlström|Göran|G|http://orcid.org/0000-0003-4981-2440', 'Duus|Jens Ø|JØ|http://orcid.org/0000-0003-3625-1250', 'Sanejouand|Yves-Henri|YH|http://orcid.org/0000-0001-8589-2666', "O'Donohue|Michael J|MJ|http://orcid.org/0000-0003-4246-3938", 'Nordberg Karlsson|Eva|E|http://orcid.org/0000-0002-8597-7050', 'Fauré|Régis|R|http://orcid.org/0000-0002-5107-9009', 'Stålbrand|Henrik|H|http://orcid.org/0000-0001-9517-6428', 'Svensson|Birte|B|http://orcid.org/0000-0002-2993-8196']
D000818:Animals D004285:Dogs D006026:Glycoside Hydrolases D006031:Glycosylation D016695:Glycosyltransferases D006868:Hydrolysis D009844:Oligosaccharides D013379:Substrate Specificity
hydrolases protein engineering transglycosylation enzyme catalysis multiple sequences alignment oligosaccharide synthesis