Detailed structural analysis of glycosidase/inhibitor interactions:: Complexes of Cex from Cellulomonas fimi with xylobiose-derived aza-sugars

Detailed insights into the mode of binding of a series of tight-binding aza-sugar glycosidase inhibitors of two fundamentally different classes are described through X-ray crystallographic studies of complexes with the retaining family 10 xylanase Cex from Cellulomonas fimi. Complexes with xylobiosederived aza-sugar inhibitors of the substituted "amidine" class (xylobio-imidazole, K-i = 150 nM; xylobiolactam oxime, K-i = 370 nM) reveal lateral interaction of the "glycosidic" nitrogen with the acid/base catalyst (Glu 127) and hydrogen bonding of the sugar 2-hydroxyl with the catalytic nucleophile (Glu233), as expected. Tight binding of xylobio-isofagomine (K-i = 130 nM) appears to be a consequence of strong interactions of the ring nitrogen with the catalytic nucleophile while, surprisingly, no direct protein contacts are made with the ring nitrogen of the xylobio-deoxynojirimycin analogue (K-i = 5800 nM). Instead the nitrogen interacts with two ordered water molecules, thereby accounting for its relatively weaker binding, though it still binds some 1200-fold more tightly than does xylobiose, presumably as a consequence of electrostatic interactions at the active site. Dramatically weaker binding of these same inhibitors to the family 11 xylanase Bcx from Bacillus circulans (K-i from 0.5 to 1.5 mM) is rationalized for the substituted amidines on the basis that this enzyme utilizes a syn protonation trajectory and likely hydrolyzes via a B-2.5 boat transition state. Weaker binding of the deoxynojirimycin and isofagomine analogues likely reflects the energetic penalty for distortion of these analogues to a B-2.5 conformation, possibly coupled with destabilizing interactions with Tyr69, a conserved, catalytically essential active site residue.