Detailed Structural Analysis of Glycosidase/Inhibitor Interactions:  Complexes of Cex from Cellulomonas fimi with Xylobiose-Derived Aza-Sugars

• Published in: Biochemistry (Easton) 2000-09, Vol.39 (38), p.11553-11563
• Main Authors: Notenboom, Valerie, Williams, Spencer J, Hoos, Roland, Withers, Stephen G, Rose, David R
• Format: Article
• Language: English
• Subjects: 1-Deoxynojirimycin - chemistry; Actinomycetales - enzymology; Aza Compounds - chemistry; aza-sugars; beta-Glucosidase - antagonists & inhibitors; beta-Glucosidase - chemistry; Cellulomonas fimi; Cex protein; Crystallization; Crystallography, X-Ray; Disaccharides - chemistry; Endo-1,4-beta Xylanases; Enzyme Inhibitors - chemistry; glycosidase; Imidazoles - chemistry; Imino Pyranoses; Lactams - chemistry; Oximes - chemistry; Piperidines - chemistry; Xylosidases - antagonists & inhibitors; Xylosidases - chemistry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0010625

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 xylobiose-derived aza-sugar inhibitors of the substituted “amidine” class (xylobio-imidazole, K i = 150 nM; xylobio-lactam oxime, K i = 370 nM) reveal lateral interaction of the “glycosidic” nitrogen with the acid/base catalyst (Glu127) 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 2,5B boat transition state. Weaker binding of the deoxynojirimycin and isofagomine analogues likely reflects the energetic penalty for distortion of these analogues to a 2,5B conformation, possibly coupled with destabilizing interactions with Tyr69, a conserved, catalytically essential active site residue.