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) [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...

How Family 26 Glycoside Hydrolases Orchestrate Catalysis on Different Polysaccharides

“…The structure of the native form of the enzyme could be traced with no breaks from Ser-7 to His-290 with the final five traceable residues from the metal affinity tag. CtLic26A forms a (␤/␣) 8 barrel structure, with the catalytic acid/base (Glu-109) and nucleophile (Glu-222) on strands ␤-4 and ␤-7, respectively (Fig. 3a), as is typical for enzymes classified into clan GH-A (2).…”

“…We have previously proposed that a crucial structural feature that helps discriminate between these two conformational pathways is the recognition of O3, an atom whose position changes markedly between 4 H 3 and B 2,5 transition states (7,8). It may be significant that the two enzyme activities specific for xylo-and gluco-configured substrates in family GH26 are both ␤-1,3 glycosidases in which O3 is tethered to the adjacent sugar which, in tandem with active-site topography, may legislate against the sugar attaining a B 2,5 transition state.…”

“…The three-dimensional structure of the enzyme has also been solved in both native and inhibited forms at 1.5 and 1.6 Å, respectively. The structures shed light on the basis for substrate specificity and also hint that although GH26 enzymes active on manno-configured substrates use a B 2,5 transition state (7,8) this is unlikely to be the case for the GH26 enzymes active on gluco-configured substrates. This work adds to the emerging picture (9) that the conformational itinerary of glycosidases is a subtle interplay of substrate configuration with enzyme three-dimensional structure, and thus, the conformational machinations are not conserved in different members of the same family.…”

“…We show that CtLic26A is a lichenase, specific for mixed (Glc␤1,4Glc␤1,4Glc␤1,3) n oligo-and polysaccharides, and displays no activity on manno-configured substrates or ␤-1,4-linked homopolymers of glucose or xylose. The three-dimensional structure of the native form of CtLic26A has been solved at 1.50-Å resolution, revealing a characteristic (␤/␣) 8 barrel with Glu-109 and Glu-222 acting as the catalytic acid/base and nucleophile in a double-displacement mechanism. The complex with the competitive inhibitor, Glc-␤-1,3-isofagomine (K i 1 M), at 1.60 Å sheds light on substrate recognition in the ؊2 and ؊1 subsites and illuminates why the enzyme is specific for lichenan-based substrates.…”

“…Thus, clan GH-A unites a large number of diversely related GHs including 1, 2, 5, 10,17,26,30,35,39,42,51,53,59,72,79, and 86 with a parallel divergence of substrate specificities (2). All these enzymes adopt the same (␤/␣) 8 fold and perform catalysis with net retention of the configuration of the anomeric carbon via a double displacement mechanism. Notably, the location of the catalytic apparatus is completely conserved, with the catalytic acid/base and nucleophile glutamates positioned on strands ␤-4 and ␤-7, respectively (2, 3).…”