Biochemical characterizations suggest that the Zn-binding site constitutes a key catalytic motif along with at least one adjacent acidic residue. Structure- and mechanism-based redesign and directed evolution have led to the emergence of Zn-dependent glycosidases with catalytic proficiency of 2.8 × 10 9 and high β-stereoselectivity. Herein, we design metalloglycosidases by constructing a hydrolytically active Zn-binding site within a barrel-shaped outer membrane protein OmpF. However, such a catalytic role of metal ions is rarely found with glycosidases that hydrolyze the glycosidic bonds in sugars. Metallohydrolases are ubiquitous in nearly all subclasses of hydrolases, utilizing metal elements to activate a water molecule and facilitate its subsequent dissociation of diverse chemical bonds.
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