Unsaturated glucuronyl hydrolase (UGL) categorized into the glycoside hydrolase family 88 catalyzes the hydrolytic release of an unsaturated glucuronic acid from glycosaminoglycan disac charides, which are produced from mammalian extracellular matrices through the β-elimination reaction of polysaccharide lyases. Here, we show enzyme characteristics of pathogenic streptococcal UGLs and structural determinants for the enzyme substrate specificity. The putative genes for UGL and phospho transferase system for amino sugar, a component of glycosam inoglycans, are assembled into a cluster in the genome of pyo genic and hemolytic streptococci such as Streptococcus agalactiae, Streptococcus pneumoniae, and Streptococcus pyo genes which produce extracellular hyaluronate lyase as a viru lent factor. The UGLs of these three streptococci were overex pressed in Escherichia coli cells, purified, and characterized. Streptococcal UGLs degraded unsaturated hyaluronate and chondroitin disaccharides most efficiently at approximately pH 5.5 and 37 °C. Distinct from Bacillus sp. GL1 UGL, streptococcal UGLs preferred sulfated substrates. DNA microarray and Western blotting indicated that the enzyme was constitutively expressed in S. agalactiae cells, although the expression level increased in the presence of glycosaminoglycan. The crystal structure of S. agalactiae UGL (SagUGL) was determined at 1.75 A resolution by xray crystallography. SagUGL adopts α[sub6]/α[sub6] barrel structure as a basic scaffold similar to Bacillus UGL, but the arrangement of amino acid residues in the active site differs between the two. SagUGL Arg236 was found to be one of the residues involved in its activity for the sulfated substrate through structural comparison and sitedirected mutagenesis. This is the first report on the structure and function of strepto coccal UGLs. [ABSTRACT FROM AUTHOR]