The ninety sequence-based families of glycoside hydrolases (GHs) [1] and the correspondingly large diversity of protein topologies [2] are a rich framework for studying variations in the catalytic mechanism of enzymatic glycoside hydrolysis. Such hydrolysis features oxocarbenium-ion-like transition states in which the anomeric centre becomes sp 2 hybridised and partial positive charge accumulates, primarily across the endocyclic O5ÀC1 bond. For pyranosides, such a species demands planarity of C5, O5, C1 and C2 at or near the transition state; a situation accommodated only by the 4 H 3 and 3 H 4 (half-chair) conformations (or their closely related envelope forms) and 2,5 B and B 2,5 boats. Initial assumptions that all glycosidases harness 4 H 3 conformations are incorrect; indeed, the utilisation of different transition states for the hydrolysis of glycosides is an emerging theme in glycobiology (Scheme 1) [3] and one that suggests a route for specific enzyme inhibition. Isofagomine lactam (1) displays an "in-plane" carbonyl at C2 and is, not surprisingly, a reasonable b-glucosidase inhibitor, with family GH1 b-glucosidases from Thermotoga maritima (TmGH1) and sweet almond inhibited with K i values of 130 nm (this work, Figure 1) and 29 mm, [4] respectively. Compound 1 has previously been shown to be an equally potent b-mannosidase inhibitor, [4] with the snail b-mannosidase inhibited with a K i of 9 mm; this is superficially extremely counter-intuitive. Here, such K i values for mannosidases are rationalised through structural analysis of 1 in complex with both an exo b-mannanase/b-mannosidase and a b-glucosidase. This work strongly supports previous proposals that b-mannosidases utilise a novel conformational itinerary, featuring a B 2,5 transition state. The ground-state axial O2 of mannose is thus pseudo-equatorial at the transition state in a way that should be harnessed in future generations of mannosidase inhibitors.Recently we described the conformational agenda of a retaining GH26 b-mannanase.[6] Trapping of the 1 S 5 conformation for the "Michaelis" complex of unhydrolysed substrate, together with the O S 2 conformation for the covalent intermediate, suggested a novel conformational itinerary for these enzymes through a B 2,5 transition state consistent with earlier proposals, notably by Sinnott [11] and Horton. [12] Glycoside hydrolases thus appear to be harnessing the full conformational itinerary in a way that is both enzyme and substrate dependent (this was recently reviewed in the context of inhibition by Vasella and colleagues).[13] Conformational considerations suggest that retaining mannosidase transition-state mimics should thus feature the pseudo-equatorial O2 of the B 2,5 conformation of mannose.Isofagomine lactam 1, synthesised by both Stick [14] and Bols, [4] contains an in-plane carbonyl at C2. In elegant work, Bols reports a K i of 9 mm for the snail b-mannosidase;[4] this inspired us to study the three-dimensional structures of 1 bound to both TmGH1 and a Cellvibrio mixtus exo b-mannanase...
