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RgNanH is an intramolecular trans-sialidase expressed by the human gut symbiont Ruminococcus gnavus, to utilise intestinal sialylated mucin glycan epitopes. Its catalytic domain, belonging to glycoside hydrolase GH33 family, cleaves off terminal sialic acid residues from mucins, releasing 2,7-anhydro-Neu5Ac which is then used as metabolic substrate by R. gnavus to proliferate in the mucosal environment. RgNanH is one of the three intramolecular trans-sialidases (IT-sialidases) characterised to date, and the first from a gut commensal organism. Here, saturation transfer difference NMR (STD NMR) in combination with computational techniques (molecular docking and CORCEMA-ST) were used to elucidate the specificity, kinetics and relative affinity of RgNanH for sialoglycans and 2,7-anhydro-Neu5Ac. We propose the first 3D model for the Michaelis complex of an IT-sialidase. This confirms the sialic acid to be the main recognition element for the interaction in the enzymatic cleft and highlights the crucial role of Trp698 to make CH-π stacking with the galactose residue of the substrate 3'-sialyllactose. The same contact is shown not to be possible for 6'-sialyllactose, due to geometrical constrains of the α-2,6 linkage. Indeed 6'-sialyllactose is not a substrate, even though it is shown to bind to RgNanH by STD NMR. These findings corroborate the role of Trp698 for the α-2,3 specificity of IT-sialidases. In this structural study, the use of Differential Epitope Mapping STD NMR (DEEP-STD NMR) approach allowed the validation of the proposed 3D models in solution. These structural approaches are shown to be instrumental in shedding light on the molecular mechanisms underpinning enzymatic reactions in the absence of enzyme-substrate X-ray structures., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)