Bacterial unsaturated glucuronyl hydrolases (UGLs) together with polysaccharide lyases are responsible for the complete depolymerization of mammalian extracellular matrix glycosaminoglycans. UGL acts on various oligosaccharides containing unsaturated glucuronic acid (Delta GlcA) at the nonreducing terminus and releases Delta GlcA through hydrolysis. In this study, we demonstrate the Substrate recognition mechanism of the UGL of Bacillus sp. GL1 by determining the X-ray crystallographic structure of its substrate-enzyme complexes. The tetrasaccharide-enzyme complex demonstrated that at least four subsites are present in the active pocket. Although several amino acid residues are crucial for substrate binding, the enzyme strongly recognizes Delta GlcA at subsite -1 through the formation of hydrogen bonds and stacking interactions, and prefers N-acetyl-D-galactosamine and glucose rather than N-acetyl-D-glucosamine as a residue accommodated in subsite +1, due to the steric hindrance. (c) 2006 Elsevier Inc. All rights reserved.