RgNanH is an intramolecular trans-sialidase (IT-sialidase) from Ruminococcus gnavus (R. gnavus), a prominent member of the gut microbiota of the healthy human gut and one of the few commensal species of bacteria which have been studied for their ability to utilise intestinal mucin glycans1. Beside, R. gnavus has been found to be highly over-represented in individuals affected by Inflammatory Bowel Disease2, an evidence that contributed to put this organism under the spot light of gut microbiota research groups in recent years.
As an IT-sialidase, RgNanH has high specificity for cleaving α2/3 sialic acid capping the mucin oligosaccharides. The hydrolysis of the terminal sialic acid is followed by intramolecular transglycosylation at the C2 position of Neu5Ac, attacked by its O7-hydroxy group at the glycerol moiety. The product of the reaction is 2,7-anhydro-Neu5Ac. Whereas the structure of the RgNanH/2,7-anhydro-Neu5Ac complex has already been solved by X-Ray diffraction3, the complexes of RgNanH and the sialoglycans Neu5Acα3Lac (3’SL) and Neu5Acα6Lac (6’SL) remain elusive.
Here we have used Saturation Transfer Difference NMR (STD NMR) and DiffErential EPitope Mapping STD NMR (DEEP-STD NMR), a novel powerful application of STD NMR developed by us4, as well as computational tools, to obtain the first 3D model representing a Michaelis complex of an IT-sialidase to date5. Besides providing an NMR validated 3D structure of the enzyme/substrate complex in solution, we also show that DEEP-STD NMR allows to gain fine structural details at the atomic level, demonstrating to be a powerful tool for the elucidation of protein-glycan complexes.