A Trapped Covalent Intermediate of a Glycoside Hydrolase on the Pathway to Transglycosylation. Insights from Experiments and Quantum Mechanics/Molecular Mechanics Simulations

• Published in: Journal of the American Chemical Society Vol. 138; no. 10; pp. 3325 - 3332
• Main Authors: Raich, Lluís, Borodkin, Vladimir, Fang, Wenxia, Castro-López, Jorge, van Aalten, Daan M. F, Hurtado-Guerrero, Ramón, Rovira, Carme
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 16.03.2016
• Subjects: Crystallography, X-Ray; Glycoside Hydrolases - chemistry; Glycoside Hydrolases - metabolism; Glycosylation; Multienzyme Complexes - chemistry; Multienzyme Complexes - metabolism; Quantum Theory; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - metabolism; Thermodynamics; Transferases - chemistry; Transferases - metabolism
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/jacs.5b10092

The conversion of glycoside hydrolases (GHs) into transglycosylases (TGs), i.e., from enzymes that hydrolyze carbohydrates to enzymes that synthesize them, represents a promising solution for the large-scale synthesis of complex carbohydrates for biotechnological purposes. However, the lack of knowledge about the molecular details of transglycosylation hampers the rational design of TGs. Here we present the first crystallographic structure of a natural glycosyl–enzyme intermediate (GEI) of Saccharomyces cerevisiae Gas2 in complex with an acceptor substrate and demonstrate, by means of quantum mechanics/molecular mechanics metadynamics simulations, that it is tuned for transglycosylation (ΔG ⧧ = 12 kcal/mol). The 2-OH···nucleophile interaction is found to be essential for catalysis: its removal raises the free energy barrier significantly (11 and 16 kcal/mol for glycosylation and transglycosylation, respectively) and alters the conformational itinerary of the substrate (from 4C1 → [4E]⧧ → 1,4B/4E to 4C1 → [4H3]⧧ → 4C1). Our results suggest that changes in the interactions involving the 2-position could have an impact on the transglycosylation activity of several GHs.