Biochemical and Structural Assessment of the 1-N-Azasugar GalNAc-isofagomine as a Potent Family 20 β-N-Acetylhexosaminidase Inhibitor

Azasugar inhibitors of the isofagomine class are potent competitive inhibitors of configuration-retaining β-glycosidases. This potency results from the formation of a strong electrostatic interaction between a protonated endocyclic nitrogen at the "anomeric" center of the inhibitor and the catalytic nucleophile of the enzyme. Although the majority of retaining β-glycosidases use a mechanism involving a carboxylate residue as a nucleophile, Streptomyces plicatus β-N-acetylhexosaminidase (SpHEX) and related family 20 glycosidases lack such a catalytic residue and use instead the carbonyl oxygen of the 2-acetamido group of the substrate as a nucleophile to "attack" the anomeric center. Thus, a strong electrostatic interaction between the inhibitor and enzyme is not expected to occur; nonetheless, the 1-N-azasugar (2R,3R,4S,5R)-2-acetamido-3,4-dihydroxy-5-hydroxymethyl-piperidinium hydrochloride (GalNAc-isofagomine·HCl), which was synthesized and assayed for its ability to inhibit SpHEX, was found to be a potent competitive inhibitor of the enzyme (K_i = 2.7 μM). A crystallographic complex of GalNAc-isofagomine bound to SpHEX was solved and refined to 1.75 Å and revealed that the lack of a strong electrostatic interaction between the "anomeric" center of GalNAc-isofagomine and SpHEX is compensated for by a novel 2.8-Å hydrogen bond formed between the equatorial proton of the endocyclic nitrogen of the azasugar ring and the carboxylate of the general acid-base residue Glu-314 of SpHEX. This interaction appears to contribute to the unexpected potency of GalNAc-isofagomine toward SpHEX.