Family 1 glycosyltransferases (GT1, UGTs) are subject to dilution-induced inactivation and low chemo stability toward their own acceptor substrates

• Published in: Frontiers in molecular biosciences Vol. 9; p. 909659
• Main Authors: Teze, David, Bidart, Gonzalo Nahuel, Welner, Ditte Hededam
• Format: Journal Article
• Language: English
• Published: Frontiers Media S.A 22.07.2022
• Subjects: biotechnology; glycosylation; glycosyltransferases; GT1; Molecular Biosciences; polyphenols; stability
• ISSN: 2296-889X; 2296-889X
• DOI: 10.3389/fmolb.2022.909659

Glycosylation reactions are essential but challenging from a conventional chemistry standpoint. Conversely, they are biotechnologically feasible as glycosyltransferases can transfer sugar to an acceptor with perfect regio- and stereo-selectivity, quantitative yields, in a single reaction and under mild conditions. Low stability is often alleged to be a limitation to the biotechnological application of glycosyltransferases. Here we show that these enzymes are not necessarily intrinsically unstable, but that they present both dilution-induced inactivation and low chemostability towards their own acceptor substrates, and that these two phenomena are synergistic. We assessed 18 distinct GT1 enzymes against three unrelated acceptors (apigenin, resveratrol, and scopoletin—respectively a flavone, a stilbene, and a coumarin), resulting in a total of 54 enzymes: substrate pairs. For each pair, we varied catalyst and acceptor concentrations to obtain 16 different reaction conditions. Fifteen of the assayed enzymes (83%) displayed both low chemostability against at least one of the assayed acceptors at submillimolar concentrations, and dilution-induced inactivation. Furthermore, sensitivity to reaction conditions seems to be related to the thermal stability of the enzymes, the three unaffected enzymes having melting temperatures above 55°C, whereas the full enzyme panel ranged from 37.4 to 61.7°C. These results are important for GT1 understanding and engineering, as well as for discovery efforts and biotechnological use.