Crystal structure of a bacterial unsaturated glucuronyl hydrolase with specificity for heparin [Glycobiology and Extracellular Matrices]

January 8th, 2014 by Nakamichi, Y., Mikami, B., Murata, K., Hashimoto, W.

Extracellular matrix molecules such as glycosaminoglycans (GAGs) are typical targets for some pathogenic bacteria, which allow adherence to host cells. Bacterial polysaccharide lyases depolymerize GAGs in β-elimination reactions, and the resulting unsaturated disaccharides are subsequently degraded to constituent monosaccharides by unsaturated glucuronyl hydrolases (UGLs). UGL substrates are classified as 1,3- and 1,4-types based on the glycoside bonds. Unsaturated chondroitin and heparin disaccharides are typical members of 1,3- and 1,4-types, respectively. Here we show the reaction modes of bacterial UGLs with unsaturated heparin disaccharides by X-ray crystallography, docking simulation, and site-directed mutagenesis. Although streptococcal and bacillus UGLs were active on unsaturated heparin disaccharides, those preferred 1,3- rather than 1,4-type substrates. The genome of GAG-degrading Pedobacter heparinus encodes 13 UGLs. Of these, Phep_2830 is known to be specific for unsaturated heparin disaccharides. The crystal structure of Phep_2830 was determined at 1.35 Å resolution. In comparison with structures of streptococcal and bacillus UGLs, a pocket-like structure and lid loop at subsite +1 are characteristic of Phep_2830. Docking simulations of Phep_2830 with unsaturated heparin disaccharides demonstrated that the direction of substrate pyranose rings differs from that in unsaturated chondroitin disaccharides. Acetyl groups of unsaturated heparin disaccharides are well accommodated in the pocket at subsite +1, and aromatic residues of the lid loop are required for stacking interactions with substrates. Thus, site-directed mutations of the pocket and lid loop led to significantly reduced enzyme activity, suggesting that the pocket-like structure and lid loop are involved in the recognition of 1,4-type substrates by UGLs.