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 A resolution with a final R-factor of 16.8% for 25 to 1.8 A 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 alpha(6)/alpha(6)-barrel, whose structure is found in the six-hairpin enzyme superfamily of an alpha/alpha-toroidal fold. One side of the UGL alpha(6)/alpha(6)-barrel structure consists of long loops containing three short beta-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 overall 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 Asp(88) and Asp(149). This was supported by site-directed mutagenesis studies in Asp(88) and Asp(149).
Highlights
Polysaccharides exist ubiquitously in nature as components of the extracellular matrix on the cell surface of many different organisms, ranging from bacteria to mammals [1]
Functional activities, and are divided into three groups: i.e. storage, e.g. starch; structural, e.g. cellulose; and functional, e.g. glycosaminoglycan. Glycosaminoglycans such as hyaluronan, chondroitin, and heparin are linear, negatively charged polysaccharides with a repeating disaccharide unit consisting of a uronic acid residue and an amino sugar residue [2]
Hyaluronan consists of D-glucuronic acid (GlcA)1 and N-acetyl-D-glucosamine (GlcNAc) (Fig. 1a) and plays an important role in cell-to-cell association in mammals and as a capsule in streptococcal bacteria [3]
Summary
GlcA, D-glucuronic acid; UGL, unsaturated glucuronyl hydrolase; DTT, dithiothreitol; MPD, 2-methyl-2,4pentanediol; GlcNAc, N-acetyl-D-glucosamine; ⌬GlcA, unsaturated Dglucuronic acid; GalNAc, N-acetyl-D-galactosamine; Glc, D-glucose; Rha, L-rhamnose; Man, D-mannose; mir, multiple isomorphous replacement; r.m.s., root mean square; AGE, N-acyl-D-glucosamine 2-epimerase. Inhibitors of polysaccharide lyases and UGL are expected to become potent pharmaceuticals for treating streptococci infectious disease. The structure provides useful information on the catalytic mechanism and for the molecular design of drugs for the treatment of streptococci, xanthomonad, and sphingomonad infectious diseases
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