Characterization of alpha-L-rhamnosidase of Bacillus sp. GL1 responsible for the complete depolymerization of gellan.

A bacterium, Bacillus sp. GL1, depolymerizes a heteropolysaccharide (gellan) to a tetrasaccharide (unsaturated glucuronyl-glucosyl-rhamnosyl-glucose) by extracellular gellan lyase. The resultant tetrasaccharide was degraded to the constituent monosaccharides by subsequent reactions of unsaturated glucuronyl hydrolase, beta-d-glucosidase, and alpha-l-rhamnosidase. alpha-l-Rhamnosidase was substantially induced in the bacterial cells when grown in a medium containing gellan as a carbon source. The purified enzyme from the cells was a monomer with a molecular mass of about 100 kDa and was most active at pH 7.0 and 50 degrees C. The enzyme acted on the gellan-degrading product (rhamnosyl-glucose) formed after successive reactions catalyzed by gellan lyase, unsaturated-glucuronyl hydrolase and beta-d-glucosidase, and released rhamnose from the disaccharide. Therefore, the alpha-l-rhamnosidase is found to be responsible as the final enzyme for the complete depolymerization of gellan.     

A novel member of glycoside hydrolase family 88: overexpression,purification, and characterization of unsaturated beta-glucuronyl hydrolase of Bacillus sp. GL1

Unsaturated beta-glucuronyl hydrolase of Bacillus sp. GL1 catalyzes the hydrolytic release of unsaturated glucuronic acids from oligosaccharides produced through the reactions of polysaccharide lyases such as gellan, xanthan, hyaluronate, and chondroitin lyases. An overexpression system for the enzyme was constructed in Escherichia coli cells involving regulation of the enzyme gene under the T7 promoter and terminator. The expression level of the enzyme in E. coli cells was 250-fold higher than that in Bacillus sp. GL1 cells. The enzyme expressed in E. coli cells was purified and characterized. The optimal pH and temperature, and substrate specificity of the purified enzyme were similar to those of the native enzyme from Bacillus sp. GL1 cells, although the enzyme expressed in E. coli cells underwent self-assembly into polymeric forms through the formation of intermolecular disulfide bonds. Circular dichroism analysis indicated that the secondary structure of the enzyme was rich in alpha-helices. Genes showing high identity (over 40% identity) with that of the enzyme were found in the genomes of some pathogenic bacteria, such as Streptococcus pyogenes and Streptococcus pneumoniae, which cause serious diseases (e.g., meningitis and pneumonia). Therefore, the enzyme of Bacillus sp. GL1 and the streptococcal proteins form a new glycoside hydrolase family, 88.     

Microbial system for polysaccharide depolymerization: enzymatic route for xanthan depolymerization by Bacillus sp. strain GL1.

An enzymatic route for the depolymerization of a heteropolysaccharide (xanthan) in Bacillus sp. strain GL1, which was closely related to Brevibacillus thermoruber, was determined by analyzing the structures of xanthan depolymerization products. The bacterium produces extracellular xanthan lyase catalyzing the cleavage of the glycosidic bond between pyruvylated mannosyl and glucuronyl residues in xanthan side chains (W. Hashimoto et al., Appl. Environ. Microbiol. 64:3765-3768, 1998). The modified xanthan after the lyase reaction was then depolymerized by extracellular beta-D-glucanase to a tetrasaccharide, without the terminal mannosyl residue of the side chain in a pentasaccharide, a repeating unit of xanthan. The tetrasaccharide was taken into cells and converted to a trisaccharide (unsaturated glucuronyl-acetylated mannosyl-glucose) by beta-D-glucosidase. The trisaccharide was then converted to the unsaturated glucuronic acid and a disaccharide (mannosyl-glucose) by unsaturated glucuronyl hydrolase. Finally, the disaccharide was hydrolyzed to mannose and glucose by alpha-D-mannosidase. This is the first complete report on xanthan depolymerization by bacteria. Novel beta-D-glucanase, one of the five enzymes involved in the depolymerization route, was purified from the culture fluid. This enzyme was a homodimer with a subunit molecular mass of 173 kDa and was most active at pH 6.0 and 45 degrees C. The enzyme specifically acted on xanthan after treatment with xanthan lyase and released the tetrasaccharide.     

A novel glycoside hydrolase family 105: the structure of family 105 unsaturated rhamnogalacturonyl hydrolase complexed with a disaccharide in comparison with family 88 enzyme complexed with the disaccharide

YteR, a hypothetical protein with unknown functions, is derived from Bacillus subtilis strain 168 and has an overall structure similar to that of bacterial unsaturated glucuronyl hydrolase (UGL), although it exhibits little amino acid sequence identity with UGL. UGL releases unsaturated glucuronic acid from glycosaminoglycan treated with glycosaminoglycan lyases. The amino acid sequence of YteR shows a significant homology (26% identity) with the hypothetical protein YesR also from B. subtilis strain 168. To clarify the intrinsic functions of YteR and YesR, both proteins were overexpressed in Escherichia coli, purified, and characterized. Based on their gene arrangements in genome and enzyme properties, YteR and YesR were found to constitute a novel enzyme activity, "unsaturated rhamnogalacturonyl hydrolase," classified as new glycoside hydrolase family 105. This enzyme acts specifically on unsaturated rhamnogalacturonan (RG) obtained from RG type-I treated with RG lyases and releases an unsaturated galacturonic acid. The crystal structure of YteR complexed with unsaturated chondroitin disaccharide (UGL substrate) was obtained and compared to the structure of UGL complexed with the same disaccharide. The UGL substrate is sterically hindered with the active pocket of YteR. The protruding loop of YteR prevents the UGL substrate from being bound effectively. The most likely candidate catalytic residues for general acid/base are Asp143 in YteR and Asp135 in YesR. This is supported by three-dimensional structural and site-directed mutagenesis studies. These findings provide molecular insights into novel enzyme catalysis and sequential reaction mechanisms involved in RG-I depolymerization by bacteria.     

Structures of Streptococcus pneumoniae hyaluronate lyase in complex with chondroitin and chondroitin sulfate disaccharides. Insights into specificity and mechanism of action

Streptococcus pneumoniae hyaluronate lyase is a surface enzyme of this Gram-positive bacterium. The enzyme degrades hyaluronan and chondroitin/chondroitin sulfates by cleaving the beta1,4-glycosidic linkage between the glycan units of these polymeric substrates. This degradation helps spreading of this bacterial organism throughout the host tissues and facilitates the disease process caused by pneumococci. The mechanism of this degradative process is based on beta-elimination, is termed proton acceptance and donation, and involves selected residues of a well defined catalytic site of the enzyme. The degradation of hyaluronan alone is thought to proceed through a processive mode of action. The structures of complexes between the enzyme and chondroitin as well as chondroitin sulfate disaccharides allowed for the first detailed insights into these interactions and the mechanism of action on chondroitins. This degradation of chondroitin/chondroitin sulfates is nonprocessive and is selective for the chondroitin sulfates only with certain sulfation patterns. Chondroitin sulfation at the 4-position on the nonreducing site of the linkage to be cleaved or 2-sulfation prevent degradation due to steric clashes with the enzyme. Evolutionary studies suggest that hyaluronate lyases evolved from chondroitin lyases and still retained chondroitin/chondroitin sulfate degradation abilities while being specialized in the degradation of hyaluronan. The more efficient processive degradation mechanism has come to be preferred for the unsulfated substrate hyaluronan.     

Crystal structures of a family 8 polysaccharide lyase reveal open and highly occluded substrate-binding cleft conformations

Bacterial enzymatic degradation of glycosaminoglycans such as hyaluronan and chondroitin is facilitated by polysaccharide lyases. Family 8 polysaccharide lyase (PL8) enzymes contain at least two domains: one predominantly composed of α-helices, the α-domain, and another predominantly composed of β-sheets, the β-domain. Simulation flexibility analyses indicate that processive exolytic cleavage of hyaluronan, by PL8 hyaluronate lyases, is likely to involve an interdomain shift, resulting in the opening/closing of the substrate-binding cleft between the α- and β-domains, facilitating substrate translocation. Here, the Streptomyces coelicolor A3(2) PL8 enzyme was recombinantly expressed in and purified from Escherichia coli and biochemically characterized as a hyaluronate lyase. By using X-ray crystallography its structure was solved in complex with hyaluronan and chondroitin disaccharides. These findings show key catalytic interactions made by the different substrates, and on comparison with all other PL8 structures reveals that the substrate-binding cleft of the S. coelicolor enzyme is highly occluded. A third structure of the enzyme, harboring a mutation of the catalytic tyrosine, created via site-directed mutagenesis, interestingly revealed an interdomain shift that resulted in the opening of the substrate-binding cleft. These results add further support to the proposed processive mechanism of action of PL8 hyaluronate lyases and may indicate that the mechanism of action is likely to be universally used by PL8 hyaluronate lyases.     

Crystal structure of Bacillus sp. GL1 xanthan lyase,which acts on the side chains of xanthan

Xanthan lyase, a member of polysaccharide lyase family 8, is a key enzyme for complete depolymerization of a bacterial heteropolysaccharide, xanthan, in Bacillus sp. GL1. The enzyme acts exolytically on the side chains of the polysaccharide. The x-ray crystallographic structure of xanthan lyase was determined by the multiple isomorphous replacement method. The crystal structures of xanthan lyase and its complex with the product (pyruvylated mannose) were refined at 2.3 and 2.4 A resolution with final R-factors of 17.5 and 16.9%, respectively. The refined structure of the product-free enzyme comprises 752 amino acid residues, 248 water molecules, and one calcium ion. The enzyme consists of N-terminal alpha-helical and C-terminal beta-sheet domains, which constitute incomplete alpha(5)/alpha(5)-barrel and anti-parallel beta-sheet structures, respectively. A deep cleft is located in the N-terminal alpha-helical domain facing the interface between the two domains. Although the overall structure of the enzyme is basically the same as that of the family 8 lyases for hyaluronate and chondroitin AC, significant differences were observed in the loop structure over the cleft. The crystal structure of the xanthan lyase complexed with pyruvylated mannose indicates that the sugar-binding site is located in the deep cleft, where aromatic and positively charged amino acid residues are involved in the binding. The Arg(313) and Tyr(315) residues in the loop from the N-terminal domain and the Arg(612) residue in the loop from the C-terminal domain directly bind to the pyruvate moiety of the product through the formation of hydrogen bonds, thus determining the substrate specificity of the enzyme.     

Purification and partial characterization of hyaluronate lyase (EC 4.2.2.1) from Propionibacterium acnes.

Hyaluronidase from Propionibacterium acnes has been purified 13,000-fold from the culture supernatant to homogeneity (as determined by polyacrylamide disc gel electrophoresis). The molecular weight of the purified enzyme was 85,110 as determined by gel filtration. The purified enzyme had a pH optimum at 6.4, was stable between pH 5 and 5.8 and was completely inactivated after 15 min at 50 degrees C. Preliminary studies suggested that the enzyme is active against chondroitin 4- and 6-sulphates, but not against dermatan sulphate. Analysis by paper chromatography of the reaction products from the degradation of hyaluronic acid by bacterial, testicular and P. acnes enzymes suggested that the P. acnes enzyme is similar in its mode of action to other bacterial hyaluronate lyases. The enzyme from P. acnes may thus be tentatively classified as a hyaluronate lyase.     

Crystal structure of unsaturated glucuronyl hydrolase complexed with substrate: molecular insights into its catalytic reaction mechanism

Unsaturated glucuronyl hydrolase (UGL), which is a member of glycoside hydrolase family GH-88, is a bacterial enzyme that degrades mammalian glycosaminoglycans and bacterial biofilms. The enzyme, which acts on unsaturated oligosaccharides with an alpha-glycoside bond produced by microbial polysaccharide lyases responsible for bacterial invasion of host cells, was believed to release 4-deoxy-l-threo-5-hexosulose-uronate (unsaturated glucuronic acid, or DeltaGlcA) and saccharide with a new nonreducing terminus by hydrolyzing the glycosidic bond. We detail the crystal structures of wild-type inactive mutant UGL of Bacillus sp. GL1 and its complex with a substrate (unsaturated chondroitin disaccharide), identify active site residues, and postulate a reaction mechanism catalyzed by UGL that triggers the hydration of the vinyl ether group in DeltaGlcA, based on the structural analysis of the enzyme-substrate complex and biochemical analysis. The proposed catalytic mechanism of UGL is a novel case among known glycosidases. Under the proposed mechanism, Asp-149 acts as a general acid and base catalyst to protonate the DeltaGlcA C4 atom and to deprotonate the water molecule. The deprotonated water molecule attacks the DeltaGlcA C5 atom to yield unstable hemiketal; this is followed by spontaneous conversion to an aldehyde (4-deoxy-l-threo-5-hexosulose-uronate) and saccharide through hemiacetal formation and cleavage of the glycosidic bond. UGL is the first clarified alpha(6)/alpha(6)-barrel enzyme using aspartic acid as the general acid/base catalyst.     

Structural determinants in streptococcal unsaturated glucuronyl hydrolase for recognition of glycosaminoglycan sulfate groups

Pathogenic Streptococcus agalactiae produces polysaccharide lyases and unsaturated glucuronyl hydrolase (UGL), which are prerequisite for complete degradation of mammalian extracellular matrices, including glycosaminoglycans such as chondroitin and hyaluronan. Unlike the Bacillus enzyme, streptococcal UGLs prefer sulfated glycosaminoglycans. Here, we show the loop flexibility for substrate binding and structural determinants for recognition of glycosaminoglycan sulfate groups in S. agalactiae UGL (SagUGL). UGL also degraded unsaturated heparin disaccharides; this indicates that the enzyme released unsaturated iduronic and glucuronic acids from substrates. We determined the crystal structures of SagUGL wild-type enzyme and both substrate-free and substrate-bound D175N mutants by x-ray crystallography and noted that the loop over the active cleft exhibits flexible motion for substrate binding. Several residues in the active cleft bind to the substrate, unsaturated chondroitin disaccharide with a sulfate group at the C-6 position of GalNAc residue. The sulfate group is hydrogen-bonded to Ser-365 and Ser-368 and close to Lys-370. As compared with wild-type enzyme, S365H, S368G, and K370I mutants exhibited higher Michaelis constants toward the substrate. The conversion of SagUGL to Bacillus sp. GL1 UGL-like enzyme via site-directed mutagenesis demonstrated that Ser-365 and Lys-370 are essential for direct binding and for electrostatic interaction, respectively, for recognition of the sulfate group by SagUGL. Molecular conversion was also achieved in SagUGL Arg-236 with an affinity for the sulfate group at the C-4 position of the GalNAc residue. These residues binding to sulfate groups are frequently conserved in pathogenic bacterial UGLs, suggesting that the motif "R-//-SXX(S)XK" (where the hyphen and slash marks in the motif indicate the presence of over 100 residues in the enzyme and parentheses indicate that Ser-368 makes little contribution to enzyme activity) is crucial for degradation of sulfated glycosaminoglycans.     

Xanthan Lyase of Bacillus sp. Strain GL1 Liberates Pyruvylated Mannose from Xanthan Side Chains

When the bacterium Bacillus sp. strain GL1 was grown in a medium containing xanthan as the carbon source, the viscosity of the medium decreased in association with growth, showing that the bacterium had xanthan-depolymerizing enzymes. One of the xanthan-depolymerizing enzymes (xanthan lyase) was present in the medium and was found to be induced by xanthan. The xanthan lyase purified from the culture fluid was a monomer with a molecular mass of 75 kDa, and was most active at pH 5.5 and 50°C. The enzyme was highly specific for xanthan and produced pyruvylated mannose. The result indicates that the enzyme cleaved the linkage between the terminal pyruvylated mannosyl and glucuronyl residues in the side chain of xanthan.     

Crystallization and some properties of chondroitinase from Arthrobacter aurescens.

A strain of Arthrobacter aurescens which secretes a large amount of chondroitinase into a culture broth, was isolated from soil. The chondroitinase was purified 380-fold over culture broth in 24% yield and crystallized. Some properties of the purified enzyme were studied and described: thermal stability (below 45 degrees), pH stability (pH 4.9 to 7.4), optimum temperature (50 degrees), and optimum pH (pH 6.0). Chrondroitin sulfate A and C, chondroitin, and hyaluronic acid were split by the enzyme but dermatan sulfate could not be. The initial rates of enzymic degradation of chondroitin sulfate C, chondroitin, and hyaluronic acid were 1.1, 1.95, and 3.2, respectively, compared to that of chondroitin sulfate A. When the enzyme was allowed to act on chondroitin sulfate A and C, the reducing power and the ultraviolet absorption at 232 nm increased proportionally to the decrease in viscosity of the substrate solution. Finally these substrates were degraded to the extent of 100% to disaccharides. By the enzyme action the main products from chondroitin sulfate A and C were deta 4,5-unsaturated disaccharides, which were identified as 2-acetamido-2-deoxy-3-O-(Beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose and 2-acet-amido-2-deoxy-3-O-(Beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose by paper chromatography, ultraviolet absorption spectroscophy, and infrared spectroscopy. Thus it is suggested that the chondroitinase is a chondroitin sulfate A and C lyase, one of the hyaluronate lyases (EC 4.2.99.1).     

Crystal structure of heparinase II from Pedobacter heparinus and its complex with a disaccharide product

Heparinase II depolymerizes heparin and heparan sulfate glycosaminoglycans, yielding unsaturated oligosaccharide products through an elimination degradation mechanism. This enzyme cleaves the oligosaccharide chain on the nonreducing end of either glucuronic or iduronic acid, sharing this characteristic with a chondroitin ABC lyase. We have determined the first structure of a heparin-degrading lyase, that of heparinase II from Pedobacter heparinus (formerly Flavobacterium heparinum), in a ligand-free state at 2.15 A resolution and in complex with a disaccharide product of heparin degradation at 2.30 A resolution. The protein is composed of three domains: an N-terminal alpha-helical domain, a central two-layered beta-sheet domain, and a C-terminal domain forming a two-layered beta-sheet. Heparinase II shows overall structural similarities to the polysaccharide lyase family 8 (PL8) enzymes chondroitin AC lyase and hyaluronate lyase. In contrast to PL8 enzymes, however, heparinase II forms stable dimers, with the two active sites formed independently within each monomer. The structure of the N-terminal domain of heparinase II is also similar to that of alginate lyases from the PL5 family. A Zn2+ ion is bound within the central domain and plays an essential structural role in the stabilization of a loop forming one wall of the substrate-binding site. The disaccharide binds in a long, deep canyon formed at the top of the N-terminal domain and by loops extending from the central domain. Based on structural comparison with the lyases from the PL5 and PL8 families having bound substrates or products, the disaccharide found in heparinase II occupies the "+1" and "+2" subsites. The structure of the enzyme-product complex, combined with data from previously characterized mutations, allows us to propose a putative chemical mechanism of heparin and heparan-sulfate degradation.     

Purification and characterization of novel chondroitin ABC and AC lyases from Bacteroides stercoris HJ-15, a human intestinal anaerobic bacterium.

Two novel chondroitinases, chondroitin ABC lyase (EC 4.2.2.4) and chondroitin AC lyase (EC 4.2.2.5), have been purified from Bacteroides stercoris HJ-15, which was isolated from human intestinal bacteria with glycosaminoglycan degrading enzymes. Chondroitin ABC lyase was purified to apparent homogeneity by a combination of QAE-cellulose, CM-Sephadex C-50, hydroxyapatite and Sephacryl S-300 column chromatography with a final specific activity of 45.7 micromol.min-1.mg-1. Chondroitin AC lyase was purified to apparent homogeneity by a combination of QAE-cellulose, CM-Sephadex C-50, hydroxyapatite and phosphocellulose column chromatography with a final specific activity of 57.03 micromol.min-1.mg-1. Chondroitin ABC lyase is a single subunit of 116 kDa by SDS/PAGE and gel filtration. Chondroitin AC lyase is composed of two identical subunits of 84 kDa by SDS/PAGE and gel filtration. Chondroitin ABC and AC lyases showed optimal activity at pH 7.0 and 40 degrees C, and 5.7-6.0 and 45-50 degrees C, respectively. Both chondroitin lyases were potently inhibited by Cu2+, Zn2+, and p-chloromercuriphenyl sulfonic acid. The purified Bacteroidal chondroitin ABC lyase acted to the greatest extent on chondroitin sulfate A (chondroitin 4-sulfate), to a lesser extent on chondroitin sulfate B (dermatan sulfate) and C (chondroitin 6-sulfate). The purified chondroitin AC lyase acted to the greatest extent on chondroitin sulfate A, and to a lesser extent on chondroitin C and hyaluronic acid. They did not act on heparin and heparan sulfate. These findings suggest that the biochemical properties of these purified chondroitin lyases are different from those of the previously purified chondroitin lyases.     

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.     

Purification and characterization of extracellular pectate lyase from Bacillus subtilis

Bacillus subtilis strain SO113 secretes a pectate lyase which is produced during the exponential death phase of growth, just before sporulation. This extracellular pectate lyase, which produces unsaturated products from polygalacturonate, was purified 35-fold from the culture supernatant of Bacillus subtilis by a CM Sephadex chromatography. It has an isoelectric point of about 9.6 and an Mr of 42,000. Optimum activity occurred at pH 8.4 and at 42 degrees C. Calcium has a stimulative effect on the enzyme activity while EDTA leads to enzyme inactivation. The pectate lyase has a specific activity of 131 mumol of aldehyde groups per min and per mg of protein. The Km of the purified enzyme for polygalacturonic acid was 0.862 g.l-1 and the Vmax for polygalacturonic acid hydrolysis was 1.475 mumol of unsaturated products per min and per mg of protein. By using monoclonal antibodies raised against Erwinia chrysanthemi 3937 pectate lyases, it was shown that pectate lyases b and c of this strain are immunologically closely related to the Bacillus subtilis pectate lyase.     

Comparison of proteins involved in chondroitin sulfate utilization by three colonic Bacteroides species.

Three species of colonic bacteria can ferment the mucopolysaccharide chondroitin sulfate: Bacteroides ovatus, Bacteroides sp. strain 3452A (an unnamed DNA homology group), and B. thetaiotaomicron. Proteins associated with the utilization of chondroitin sulfate by B. thetaiotaomicron have been characterized previously. In this report we compare chondroitin lyases and chondroitin sulfate-associated outer membrane polypeptides of B. ovatus and Bacteroides sp. strain 3452A with those of B. thetaiotaomicron. All three species produce two soluble cell-associated chondroitin lyases, chondroitin lyase I and II. Purified enzymes from the three species have similar pH optima, Km values, and molecular weights. However, peptide mapping experiments show that the chondroitin lyases from B. ovatus and Bacteroides sp. strain 3452A are not identical to those of B. thetaiotaomicron. A cloned gene that codes for the chondroitin lyase II from B. thetaiotaomicron hybridized on a Southern blot with DNA from B. ovatus or Bacteroides sp. strain 3452A only when low-stringency conditions were used. Antibody to chondroitin lyase II from B. thetaiotaomicron did not cross-react with chondroitin lyase II from B. ovatus or Bacteroides sp. strain 3452A. Chondroitin lyase activity in all three species was inducible by chondroitin sulfate. B. ovatus and Bacteroides sp. strain 3452A, like B. thetaiotaomicron, have outer membrane polypeptides that appear to be regulated by chondroitin sulfate, but the chondroitin sulfate-associated outer membrane polypeptides differ in molecular weight. Despite these differences, the ability of intact bacteria to utilize chondroitin sulfate, as indicated by growth yields in carbohydrate-limited continuous culture and the rate at which the chondroitin lyases were induced, was the same for all three species.     

Comparison of proteins involved in chondroitin sulfate utilization by three colonic Bacteroides species

Three species of colonic bacteria can ferment the mucopolysaccharide chondroitin sulfate: Bacteroides ovatus, Bacteroides sp. strain 3452A (an unnamed DNA homology group), and B. thetaiotaomicron. Proteins associated with the utilization of chondroitin sulfate by B. thetaiotaomicron have been characterized previously. In this report we compare chondroitin lyases and chondroitin sulfate-associated outer membrane polypeptides of B. ovatus and Bacteroides sp. strain 3452A with those of B. thetaiotaomicron. All three species produce two soluble cell-associated chondroitin lyases, chondroitin lyase I and II. Purified enzymes from the three species have similar pH optima, Km values, and molecular weights. However, peptide mapping experiments show that the chondroitin lyases from B. ovatus and Bacteroides sp. strain 3452A are not identical to those of B. thetaiotaomicron. A cloned gene that codes for the chondroitin lyase II from B. thetaiotaomicron hybridized on a Southern blot with DNA from B. ovatus or Bacteroides sp. strain 3452A only when low-stringency conditions were used. Antibody to chondroitin lyase II from B. thetaiotaomicron did not cross-react with chondroitin lyase II from B. ovatus or Bacteroides sp. strain 3452A. Chondroitin lyase activity in all three species was inducible by chondroitin sulfate. B. ovatus and Bacteroides sp. strain 3452A, like B. thetaiotaomicron, have outer membrane polypeptides that appear to be regulated by chondroitin sulfate, but the chondroitin sulfate-associated outer membrane polypeptides differ in molecular weight. Despite these differences, the ability of intact bacteria to utilize chondroitin sulfate, as indicated by growth yields in carbohydrate-limited continuous culture and the rate at which the chondroitin lyases were induced, was the same for all three species.     

Cartilage degradation by hyaluronate lyase and chondroitin ABC lyase: a MALDI-TOF mass spectrometric study

Matrix-assisted laser desorption ionization and time-of-flight mass spectrometry (MALDI-TOF MS) has been used to investigate degradation products of two selected polysaccharides of cartilage (chondroitin sulfate and hyaluronic acid). Testicular hyaluronate lyase and chondroitin ABC lyase were used for enzymic digestion of both polysaccharides as well as of cartilage specimens. Polysaccharide solutions and cartilage supernatants were assayed by positive and negative MALDI-TOF MS. Especially chondroitin ABC lyase produced high amounts of digestion products (unsaturated di- and tetrasaccharides) from polysaccharides as well as from cartilage, clearly monitored by MALDI-TOF MS. It is concluded that MALDI-TOF MS provides a precise and fast tool for the determination of oligosaccharides since no previous derivatization is required.