Abstract
Unsaturated glucuronyl hydrolase (UGL) categorized into the glycoside hydrolase family 88 catalyzes the hydrolytic release of an unsaturated glucuronic acid from glycosaminoglycan disaccharides, which are produced from mammalian extracellular matrices through the beta-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 phosphotransferase system for amino sugar, a component of glycosaminoglycans, are assembled into a cluster in the genome of pyogenic and hemolytic streptococci such as Streptococcus agalactiae, Streptococcus pneumoniae, and Streptococcus pyogenes, which produce extracellular hyaluronate lyase as a virulent factor. The UGLs of these three streptococci were overexpressed in Escherichia coli cells, purified, and characterized. Streptococcal UGLs degraded unsaturated hyaluronate and chondroitin disaccharides most efficiently at approximately pH 5.5 and 37 degrees 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 x-ray crystallography. SagUGL adopts alpha(6)/alpha(6)-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 Arg-236 was found to be one of the residues involved in its activity for the sulfated substrate through structural comparison and site-directed mutagenesis. This is the first report on the structure and function of streptococcal UGLs.
Highlights
Cell surface polysaccharides play an important role in linking neighboring cells and protecting cells against physicochemical stress such as osmotic pressure or invasion by pathogens
GL1 Unsaturated glucuronyl hydrolase (UGL); ⌬6S, unsaturated chondroitin disaccharide sulfated at C-6 position of GalNAc residue; SagUGL, S. agalactiae UGL; SpnUGL, S. pneumoniae UGL; SpyUGL, S. pyogenes UGL; GFC, gel filtration chromatography; ⌬4S, unsaturated chondroitin disaccharide sulfated at C-4 position of GalNAc residue; MES, 4-morpholineethanesulfonic acid
In S. pneumoniae, the gene for UGL homolog is located near the hyaluronate lyase gene in the genome [10] (Fig. 2A), suggesting that the two proteins are cooperatively involved in degrading glycosaminoglycans
Summary
Homology and Sequence Alignment Analysis—To find novel proteins homologous to Bacillus sp. GL1 UGL (BacillusUGL), the BLAST and CLUSTALW programs were used to search for a sequence similarity and for multiple sequence alignment, respectively. TLC was performed to investigate the enzyme activity using a high concentration of substrate and enzyme as described previously [23]. Absorbance coefficients of S. agalactiae UGL (SagUGL), S. pneumoniae UGL (SpnUGL), and S. pyogenes UGL (SpyUGL) are 2.30, 2.15, and 2.21 for 1 mg/ml protein, respectively. To clone the streptococcal UGL genes, PCR was conducted in a reaction mixture (0.1 ml) consisting of 5 units KOD polymerase (Toyobo), 0.25 g of genomic DNA, 40 pmol of forward and reverse primers, 20 nmol of dNTPs, 100 nmol of MgCl2, 5 l of dimethyl sulfoxide, and the commercial reaction buffer supplied with KOD polymerase. Nucleotide sequences of the PCR products were confirmed to completely match those of streptococcal UGL genes by DNA sequencing. The PCR products were ligated with HincII-digested pUC119 (Takara Bio), and the resultant plasmids were digested with
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