The conformational changes in a sugar moiety along the hydrolytic pathway are key to understand the mechanism of glycoside hydrolases (GHs) and to design new inhibitors. The two predominant itineraries for mannosidases go via O S2 →B2,5 →1 S5 and 3 S1 →3 H4 →1 C4 . For the CAZy family 92, the conformational itinerary was unknown. Published complexes of Bacteroides thetaiotaomicron GH92 catalyst with a S-glycoside and mannoimidazole indicate a 4 C1 →4 H5 /1 S5 →1 S5 mechanism. However, as observed with the GH125 family, S-glycosides may not act always as good mimics of GH's natural substrate. Here we present a cooperative study between computations and experiments where our results predict the E5 →B2,5 /1 S5 →1 S5 pathway for GH92 enzymes. Furthermore, we demonstrate the Michaelis complex mimicry of a new kind of C-disaccharides, whose biochemical applicability was still a chimera.

Department of Chemistry of Natural Compounds University of Chemistry and Technology Technická 5 166 28 Prague Czech Republic

Department of Structural and Computational Biology Max F Perutz Laboratories University of Vienna Dr Bohr Gasse 9 1030 Vienna Austria

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Gilead Sciences and IOCB Research Centre Czech Academy of Sciences Flemingovo nám 2 166 10 Prague Czech Republic

Research Institute of Green Science and Technology Shizuoka University 836 Ohya Suruga ku Shizuoka 422 8529 Japan

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