Thioglycoligase derived from fungal GH3 β-xylosidase is a multi-glycoligase with broad acceptor tolerance

• Published in: Nature communications Vol. 11; no. 1; pp. 4864 - 16
• Main Authors: Nieto-Domínguez, Manuel, Fernández de Toro, Beatriz, de Eugenio, Laura I., Santana, Andrés G., Bejarano-Muñoz, Lara, Armstrong, Zach, Méndez-Líter, Juan Antonio, Asensio, Juan Luis, Prieto, Alicia, Withers, Stephen G., Cañada, Francisco Javier, Martínez, María Jesús
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 25.09.2020; Nature Portfolio
• Subjects: 140/131; 140/58; 631/45/221; 631/45/603; 631/45/607/1164; 631/61/168; 631/92/603; 82/83; Biocatalysis; Catalytic Domain; Drug Tolerance - physiology; Fungi - drug effects; Fungi - enzymology; Fungi - metabolism; Glycoconjugates - metabolism; Glycoside Hydrolases - metabolism; Glycosides - chemistry; Glycosylation; Humanities and Social Sciences; Hydrogen-Ion Concentration; Kinetics; Models, Molecular; multidisciplinary; Mutagenesis; Science; Science (multidisciplinary); Substrate Specificity; Talaromyces - enzymology; Talaromyces - genetics; Xylosidases - chemistry; Xylosidases - genetics; Xylosidases - metabolism
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-020-18667-3

The synthesis of customized glycoconjugates constitutes a major goal for biocatalysis. To this end, engineered glycosidases have received great attention and, among them, thioglycoligases have proved useful to connect carbohydrates to non-sugar acceptors. However, hitherto the scope of these biocatalysts was considered limited to strong nucleophilic acceptors. Based on the particularities of the GH3 glycosidase family active site, we hypothesized that converting a suitable member into a thioglycoligase could boost the acceptor range. Herein we show the engineering of an acidophilic fungal β-xylosidase into a thioglycoligase with broad acceptor promiscuity. The mutant enzyme displays the ability to form O-, N-, S- and Se- glycosides together with sugar esters and phosphoesters with conversion yields from moderate to high. Analyses also indicate that the p K a of the target compound was the main factor to determine its suitability as glycosylation acceptor. These results expand on the glycoconjugate portfolio attainable through biocatalysis. Thioglycoligases have proved useful for bonding carbohydrates to non-sugar acceptors, however, the scope of these biocatalysts is usually limited. Here, the authors engineer a xylosidase into a thioglycoligase with the ability to form O-, N-, S- and Se- glycosides together with sugar esters and phosphoesters.