Common Inhibition of Both β‐Glucosidases and β‐Mannosidases by Isofagomine Lactam Reflects Different Conformational Itineraries for Pyranoside Hydrolysis

Abstract

The ninety sequence-based families of glycoside hydrolases (GHs) and the correspondingly large diversity of protein topologies 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 hybridised and partial positive charge accumulates, primarily across the endocyclic O5 C1 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 H3 and H4 (half-chair) conformations (or their closely related envelope forms) and B and B2,5 boats. Initial assumptions that all glycosidases harness H3 conformations are incorrect; indeed, the utilisation of different transition states for the hydrolysis of glycosides is an emerging theme in glycobiology (Scheme 1) 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 Ki values of 130 nm (this work, Figure 1) and 29 mm, respectively. Compound 1 has previously been shown to be an equally potent b-mannosidase inhibitor, with the snail b-mannosidase inhibited with a Ki of 9 mm ; this is superficially extremely counter-intuitive. Here, such Ki 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 B2,5 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. Trapping of the S5 conformation for the “Michaelis” complex of unhydrolysed substrate, together with the S2 conformation for the covalent intermediate, suggested a novel conformational itinerary for these enzymes through a B2,5 transition state consistent with earlier proposals, notably by Sinnott and Horton. 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). Conformational considerations suggest that retaining mannosidase transition-state mimics should thus feature the pseudo-equatorial O2 of the B2,5 conformation of mannose. Isofagomine lactam 1, synthesised by both Stick and Bols, contains an in-plane carbonyl at C2. In elegant work, Bols reports a Ki 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 (CmMan5, which is totally specific for manno-configured substrates). “Dual” inhibition of b-glucosidases and b-mannosidases has also been reported for the hydropyridazone compounds, which show similarity to isofagomine lactam, as discussed below. The three-dimensional structures of TmGH1 and CmMan5 in complex with 1 were determined by X-ray [a] Dr. F. Vincent, T. M. Gloster, Prof. G. J. Davies Structural Biology Laboratory, Department of Chemistry The University of York, Heslington, York, YO10 5YW (UK). Fax: (+44)1904-328266 E-mail : davies@ysbl.york.ac.uk