Combined Inhibitor Free‐Energy Landscape and Structural Analysis Reports on the Mannosidase Conformational Coordinate

Abstract

Mannosidases catalyze the hydrolysis of a diverse range of polysaccharides and glycoconjugates, and the various sequence-based mannosidase families have evolved ingenious strategies to overcome the stereoelectronic challenges of mannoside chemistry. Using a combination of computational chemistry, inhibitor design and synthesis, and X-ray crystallography of inhibitor/enzyme complexes, it is demonstrated that mannoimidazole-type inhibitors are energetically poised to report faithfully on mannosidase transition-state conformation, and provide direct evidence for the conformational itinerary used by diverse mannosidases, including b-mannanases from families GH26 and GH113. Isofagomine-type inhibitors are poor mimics of transition-state conformation, owing to the high energy barriers that must be crossed to attain mechanistically relevant conformations, however, these sugarshaped heterocycles allow the acquisition of ternary complexes that span the active site, thus providing valuable insight into active-site residues involved in substrate recognition.

Dissecting conformational contributions to glycosidase catalysis and inhibition

“…While superficially these compounds resemble aspects of the proposed transition state, there are intrinsic limitations of what can be mimicked in a chemically stable compound, including hybridization changes, partial charge development, and fractional bond orders. A study of the FELs of two inhibitors that display superficial transition state mimicry: isofagomine and mannoimidazole revealed dramatic differences (Figure 2a) [30 •• ]. Isofagomine is strongly biased toward a 4 C 1 conformation, with potential transition state mimicking 4 H 3 and B 2,5 conformations lying 12 and 8 kcal mol −1 higher, respectively, and importantly with a significant barrier to attaining those conformations.…”

“…There is now compelling evidence that family GH2, 26 and 113 retaining β-mannosidases, and GH38 retaining and GH92 inverting α-mannosidases, utilize B 2,5 transition states with Michaelis complexes in a 1 S 5 (for β-) or O S 2 (for α-), and thus operate through 1 S 5 ↔ B 2,5 ‡ ↔ O S 2 conformational itineraries [30 •• ]. The Michaelis complex conformation provides a pseudo axial arrangement of the anomeric leaving group and permits inline attack of the nucleophile; and importantly the 1 S 5 conformation relieves the Δ2 effect.…”

Tailoring recombinant protein quality by rational media design

“…This observation highlights the pivotal role of Ca 2+ in the catalytic cleavage of α-1,2 bonds 98 . In addition to kifunensine other inhibitors such as swainsonine and mannoimidazole inhibit mannosidases as well 98,99 Mannostatin A, a reversible and competitive inhibitor of Golgi α-mannosidase II, belongs to the most potent inhibitors of this enzyme 100,101 . Zinc plays an important catalytic role in the hydrolyzation step involving α-mannosidase II, as it helps to stabilize the transition state by relieving the electron deficiency of the Michaelis complex 102 .…”

Structural Basis of Specific Glucoimidazole and Mannoimidazole Binding by Os3BGlu7

“…One powerful strategy to assign conformational itineraries or describe how molecules change shape along a reaction coordinate is the use of TS analogue inhibitors [ 27 ]. In fact, the investigation of TS conformation is useful for predicting conformational itineraries (from reactant to product state via the TS) based on the principle that the reaction pathway with the least nuclear motion, i.e., the least change in atomic position and electronic configuration will be favored [ 28 ].…”