Crystal structure of unsaturated glucuronyl hydrolase, responsible for the degradation of glycosaminoglycan, from Bacillus sp. GL1 at 1.8 Å resolution

Abstract

Unsaturated glucuronyl hydrolase (UGL) is a novel glycosaminoglycan hydrolase that releases unsaturated D-glucuronic acid from oligosaccharides produced by polysaccharide lyases. The x-ray crystallographic structure of UGL from Bacillus sp. GL1 was first determined by multiple isomorphous replacement (mir) and refined at 1.8 Å resolution with a final R-factor of 16.8% for 25 to 1.8 Å resolution data. The refined UGL structure consists of 377 amino acid residues and 478 water molecules, four glycine molecules, two dithiothreitol (DTT) molecules, and one 2-methyl-2,4-pentanediol (MPD) molecule. UGL includes an α6/α6-barrel, whose structure is found in the six-hairpin enzyme superfamily of an α/α-toroidal fold. One side of the UGL α6/α6-barrel structure consists of long loops containing three short β-sheets and contributes to the formation of a deep pocket. One glycine molecule and two DTT molecules surrounded by highly conserved amino acid residues in UGLs were found in the pocket, suggesting that catalytic and substrate-binding sites are located in this pocket. The over-all UGL structure, with the exception of some loops, very much resembled that of the Bacillus subtilis hypothetical protein Yter, whose function is unknown and which exhibits little amino acid sequence identity with UGL. In the active pocket, residues possibly involved in substrate recognition and catalysis by UGL are conserved in UGLs and Yter. The most likely candidate catalytic residues for glycosyl hydrolysis are Aspss and Asp149. This was supported by site-directed mutagenesis studies in Asp88 and Asp149.