The Structural Basis of the Inhibition of Golgi α-Mannosidase II by Mannostatin A and the Role of the Thiomethyl Moiety in Ligand-Protein Interactions

摘要

The X-ray crystal structures of mannose trimming enzyme Drosophila Golgi α–mannosidase II (dGMII) complexed with the inhibitors mannostatin A (>1) and an N-benzyl analog (>2) have been determined. Molecular dynamics simulations and NMR studies have shown that the five-membered ring of mannostatin A is rather flexible occupying pseudo-rotational itineraries between ²T3 and ⁵E, and ²T3 and ⁴E. In the bound state, mannostatin A adopts a ²T1 twist envelope conformation, which is not significantly populated in solution. Possible conformations of the mannosyl oxacarbenium ion and an enzyme-linked intermediate have been compared to the conformation of mannostatin A in the co-crystal structure with dGMII. It has been found that mannostatin A best mimics the covalent linked mannosyl intermediate, which adopts a ¹S5 skew boat conformation. The thiomethyl group, which is critical for high affinity, superimposes with the C-6 hydroxyl of the covalent linked intermediate. This functionality is able to make a number of additional polar and non-polar interactions increasing the affinity for dGMII. Furthermore, the X-ray structures show that the environment surrounding the thiomethyl group of >1 is remarkably similar to the arrangements around the methionine residues in the protein. Collectively, our studies contradict the long held view that potent inhibitors of glycosidases mimic an oxacarbenium ion like transition state.