Substrate Specificity of Streptococcal Unsaturated Glucuronyl Hydrolases for Sulfated Glycosaminoglycan

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 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 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 °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 Å resolution by x-ray crystallography. SagUGL adopts α₆/α₆-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.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. Glycosaminoglycans such as chondroitin, hyaluronan, and heparin are highly negatively charged polysaccharides with a repeating disaccharide unit consisting of an uronic acid residue (glucuronic or iduronic acid) and an amino sugar residue (glucosamine or galactosamine) (1), and they are widely present in mammalian cells as an extracellular matrix responsible for cell-to-cell association, cell signaling, and cell growth and differentiation (2). For example, in humans, glycosaminoglycans exist in tissues such as the eye, brain, liver, skin, and blood (3). Except for hyaluronan, glycosaminoglycans such as chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin sulfate, and heparan sulfate are often sulfated. Chondroitin consists of d-glucuronic acid (GlcA)² and N-acetyl-d-galactosamine (GalNAc) with a sulfate group(s) at position 4 or 6 or both (4). Hyaluronan, is composed of GlcA and N-acetyl-d-glucosamine (GlcNAc) (5).The adhesion of pathogenic bacteria to mammalian cells is regarded as a primary mechanism of bacterial infection, followed by secondary effects of the infectious process. Polysaccharides, including the glycosaminoglycans that form part of the cell surface matrix, are typical targets for microbial pathogens that invade host cells, and many specific interactions between pathogens and these polysaccharides have been described (6). Glycosaminoglycans in the extracellular matrix are also degraded enzymatically by hydrolases and lyases (1). Generally, hydrolases cleave the glycoside bonds between the glycosyl oxygen and the anomeric carbon atom through the addition of water and play an important role in glycosaminoglycan metabolism in mammals (7). On the other hand, bacterial pathogens invading host cells degrade glycosaminoglycans through the action of lyases. Bacterial polysaccharide lyases recognize the uronic acid residue in polysaccharides, cleave the glycoside bonds through the β-elimination reaction without water addition, and produce unsaturated saccharides with the unsaturated uronic acid residue having a CC double bond at the nonreducing terminus (8).Streptococci such as group B Streptococcus agalactiae, group nonassigned Streptococcus pneumoniae, and group A Streptococcus pyogenes are typical pyogenic and hemolytic pathogens causing severe infections (e.g. pneumonia, bacteremia, sinusitis, or meningitis) (9–11). In S. pneumoniae, hyaluronate lyase, neuraminidases, autolysin, choline-binding protein A, and pneumococcal surface protein A are suggested to function as cell surface virulent factors (12). Hyaluronate lyase degrades the extracellular matrix component hyaluronan in mammalian cells through the β-elimination reaction and releases unsaturated disaccharide, indicating that the enzyme produced by pathogenic bacteria functions as a spreading factor (13). Because hyaluronate lyase is commonly produced by the three pyogenic and hemolytic streptococci (14–16), the structure and function of their enzymes have been intensively studied (17, 18). Groups A, B, C, and G streptococci also produce hyaluronate lyase (19), suggesting that the enzyme is ubiquitously present in pathogenic streptococci. Streptococcal hyaluronate lyase can also act on sulfated and nonsulfated chondroitin (20). The metabolism of the resultant unsaturated disaccharides in streptococci, however, remains to be clarified.Unsaturated glucuronyl hydrolase (UGL), a member of the glycoside hydrolase family 88 in the CAZY data base (21), acts on unsaturated oligosaccharides having an unsaturated GlcA (ΔGlcA) with β-glycoside bond, such as ΔGlcA-GalNAc produced by chondroitin lyase and ΔGlcA-GlcNAc produced by hyaluronate lyase (22) (Fig. 1A). We have first identified the UGL-coding gene in Bacillus sp. GL1 (23) and clarified the structure and function of the enzyme by x-ray crystallography (24–27). The enzyme reaction generates ΔGlcA and the leaving saccharide. ΔGlcA is spontaneously converted to 4-deoxy-1-threo-5-hexosulose-uronate (Fig. 1A) because the ringed form of ΔGlcA has not been obtained because of keto-enole equilibrium (23, 28). In contrast with general glycoside hydrolases with retention or inversion catalytic mechanism of an anomeric configuration, UGL uniquely triggers hydrolysis of vinyl ether groups in unsaturated saccharides but not of the glycoside bond (26) (Fig. 1B). This article deals with the characteristics of streptococcal UGLs by using recombinant enzymes, gene expression in S. agalactiae cells by DNA microarray, and structural determinants of S. agalactiae UGL for substrate specificity by x-ray crystallography and site-directed mutagenesis.Open in a separate windowFIGURE 1.UGL reaction. A, degradation scheme of Δ6S by UGL. B, catalytic reaction mechanism of UGL. C, structures of unsaturated oligosaccharides. ΔGellan, unsaturated gellan tetrasaccharide; ΔHA, unsaturated hyaluronan disaccharide; Δ0S, unsaturated chondroitin disaccharide; Δ2′S, unsaturated chondroitin disaccharide sulfated at C-2 position of ΔGlcA residue; Δ2′S4S, unsaturated chondroitin disaccharide sulfated at C-2 position of ΔGlcA residue and C-4 position of GalNAc residue; Δ2′S6S, unsaturated chondroitin disaccharide sulfated at C-2 position of ΔGlcA residue and C-6 position of GalNAc residue; Δ4S6S, unsaturated chondroitin disaccharide sulfated at C-4 and C-6 positions of GalNAc residue; Δ2′S4S6S, unsaturated chondroitin disaccharide sulfated at C-2 position of ΔGlcA residue and C-4 and C-6 positions of GalNAc residue.