Crystal structure of unsaturated glucuronyl hydrolase complexed with substrate

Unsaturated glucuronyl hydrolase (UGL), which is a member of glycoside hydrolase family GH- 88, is a bacterial enzyme that degrades mammalian glycosaminoglycans and bacterial biofilms. The enzyme, which acts on unsaturated oligosaccharides with an alpha- glycoside bond produced by microbial polysaccharide lyases responsible for bacterial invasion of host cells, was believed to release 4- deoxy- L- threo- 5- hexosulose- uronate ( unsaturated glucuronic acid, or Delta GlcA) and saccharide with a new nonreducing terminus by hydrolyzing the glycosidic bond. We detail the crystal structures of wild-type inactive mutant UGLof Bacillus sp. GL1 and its complex with a substrate ( unsaturated chondroitin disaccharide), identify active site residues, and postulate a reaction mechanism catalyzed by UGL that triggers the hydration of the vinyl ether group in Delta GlcA, based on the structural analysis of the enzyme- substrate complex and biochemical analysis. The proposed catalytic mechanism of UGL is a novel case among known glycosidases. Under the proposed mechanism, Asp- 149 acts as a general acid and base catalyst to protonate the Delta GlcA C4 atom and to deprotonate the water molecule. The deprotonated water molecule attacks the Delta GlcA C5 atom to yield unstable hemiketal; this is followed by spontaneous conversion to an aldehyde ( 4- deoxy- L- threo- 5-hexosulose- uronate) and saccharide through hemiacetal formation and cleavage of the glycosidic bond. UGL is the first clarified alpha(6)/alpha(6)- barrel enzyme using aspartic acid as the general acid/ base catalyst.