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• W1990636799 abstract "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. 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. Glycosaminoglycans (e.g. hyaluronan, chondroitin, and heparin) are mammalian extracellular matrix polysaccharides with a repeating disaccharide unit that consists of a uronic acid residue, such as d-glucuronic acid (GlcUA) 2The abbreviations used are GlcUA, d-glucuronic acid; IdoUA, l-iduronic acid; GlcN, d-glucosamine; GlcNAc, N-acetyl-d-glucosamine; UGL, unsaturated glucuronyl hydrolase; ΔGlcUA, unsaturated GlcUA; BacillusUGL, Bacillus sp. GL1 UGL; Δ6S, unsaturated chondroitin disaccharide sulfated at C-6 position of GalNAc residue; SagUGL, S. agalactiae UGL; WT, wild type; D175N, SagUGL mutant with Asp-175 substituted with Asn; ΔIdoUA, unsaturated iduronic acid; T235A, SagUGL mutant with Thr-235 substituted with Ala; R236H, SagUGL mutant with Arg-236 substituted with His; S365H, SagUGL mutant with Ser-365 substituted with His; S368G, SagUGL mutant with Ser-368 substituted with Gly; K370I, SagUGL mutant with Lys-370 substituted with Ile; H210R, BacillusUGL mutant with His-210 substituted with Arg; H339S, BacillusUGL mutant with His-339 substituted with Ser; G342S, BacillusUGL mutant with Gly-342 substituted with Ser; I344K, BacillusUGL mutant with Ile-344 substituted with Lys; Δ0S, unsaturated sulfate-free chondroitin disaccharide; Δ4S, unsaturated chondroitin disaccharide sulfated at C-4 position of GalNAc residue. or l-iduronic acid (IdoUA), and an amino sugar residue, such as d-glucosamine (GlcN), N-acetyl-d-glucosamine (GlcNAc), or N-acetyl-d-galactosamine (GalNAc) (1Ernst S. Langer R. Cooney C.L. Sasisekharan R. Crit. Rev. Biochem. Mol. Biol. 1995; 30: 387-444Crossref PubMed Scopus (352) Google Scholar, 2Hascall V. Esko J.D. 2nd Ed. Essentials of Glycobiology. Vol. 15. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2009: 219-228Google Scholar). These extracellular matrices, which are present in various mammalian tissues, play an important role in cell signaling, growth and differentiation, and cell-to-cell association to maintain the architecture of connective tissues (3Gandhi N.S. Mancera R.L. Chem. Biol. Drug Des. 2008; 72: 455-482Crossref PubMed Scopus (749) Google Scholar). A large number of glycosaminoglycans, with the exception of hyaluronan, are frequently sulfated (4Lindahl U. Höök M. Annu. Rev. Biochem. 1978; 47: 385-417Crossref PubMed Scopus (576) Google Scholar), and the sulfate groups together with uronic acids enhance the negative charge of the polysaccharides. For instance, chondroitin has sulfate group(s) at the C-4 and/or C-6 positions of the GalNAc residue and/or at the C-2 position of the GlcUA residue (5Lauder R.M. Complement. Ther. Med. 2009; 17: 56-62Crossref PubMed Scopus (126) Google Scholar). Degradation of glycosaminoglycans has been studied previously from the viewpoint of bacterial infections and neural regenerations. Bacterial pathogens, such as streptococci, degrade glycosaminoglycans to invade host cells by producing polysaccharide lyases (6Jedrzejas M.J. Cell. Mol. Life Sci. 2007; 64: 2799-2822Crossref PubMed Scopus (44) Google Scholar). Distinct from polysaccharide hydrolases, glycosaminoglycan lyases recognize the uronic acid residue, cleave the linkage between sugars through the β-elimination reaction, and produce unsaturated oligosaccharides, resulting in the unsaturated uronic acid residue having a C=C double bond at the nonreducing terminus (7Linhardt R.J. Avci F.Y. Toida T. Kim Y.S. Cygler M. Adv. Pharmacol. 2006; 53: 187-215Crossref PubMed Scopus (51) Google Scholar) (see Fig. 1A). Many pathogenic streptococci produce hyaluronate lyases as a spreading factor; these enzymes are capable of degrading both sulfated and unsulfated chondroitin as well as hyaluronan (8Paton J.C. Andrew P.W. Boulnois G.J. Mitchell T.J. Annu. Rev. Microbiol. 1993; 47: 89-115Crossref PubMed Scopus (205) Google Scholar, 9Gase K. Ozegowski J. Malke H. Biochim. Biophys. Acta. 1998; 1398: 86-98Crossref PubMed Scopus (35) Google Scholar, 10Berry A.M. Lock R.A. Thomas S.M. Rajan D.P. Hansman D. Paton J.C. Infect. Immun. 1994; 62: 1101-1118Crossref PubMed Google Scholar, 11Hynes W.L. Dixon A.R. Walton S.L. Aridgides L.J. FEMS Microbiol. Lett. 2000; 184: 109-112Crossref PubMed Google Scholar, 12Pritchard D.G. Lin B. Willingham T.R. Baker J.R. Arch. Biochem. Biophys. 1994; 315: 431-437Crossref PubMed Scopus (79) Google Scholar). Researchers have extensively studied the structure and function of streptococcal hyaluronate lyases to ultimately facilitate the development of therapeutic agents for inhibition of the lyases (13Li S. Kelly S.J. Lamani E. Ferraroni M. Jedrzejas M.J. EMBO J. 2000; 19: 1228-1240Crossref PubMed Scopus (149) Google Scholar, 14Mello L.V. De Groot B.L. Li S. Jedrzejas M.J. J. Biol. Chem. 2002; 277: 36678-36688Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). On the other hand, bacterial polysaccharide lyases (e.g. chondroitin lyase ABC) have been demonstrated to promote neural regeneration (15Bradbury E.J. Moon L.D. Popat R.J. King V.R. Bennett G.S. Patel P.N. Fawcett J.W. McMahon S.B. Nature. 2002; 416: 636-640Crossref PubMed Scopus (1917) Google Scholar). Neurons, especially their axons, experience difficulty in regeneration due to the presence of some inhibitory molecules, such as chondroitin sulfate proteoglycans (16Kwok J.C. Afshari F. García-Alías G. Fawcett J.W. Restor. Neurol. Neurosci. 2008; 26: 131-145PubMed Google Scholar). Enzymatic degradation of chondroitin sulfate at the site of an injury enables neurons to regenerate axons and to restore postsynaptic activity. Therefore, elucidation of the bacterial mechanism underlying glycosaminoglycan degradation is important for establishment of therapy against bacterial infections and neural injury. Unsaturated glucuronyl hydrolase (UGL) acts on unsaturated glycosaminoglycan oligosaccharides that are produced by polysaccharide lyases and catalyzes the hydrolytic release of unsaturated GlcUA (ΔGlcUA) from saccharides (17Itoh T. Akao S. Hashimoto W. Mikami B. Murata K. J. Biol. Chem. 2004; 279: 31804-31812Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) (see Fig. 1A). UGL is peculiar among general glycoside hydrolases in that it triggers hydration of vinyl ether groups specifically present in unsaturated saccharides but not of glycoside bonds (18Itoh T. Hashimoto W. Mikami B. Murata K. J. Biol. Chem. 2006; 281: 29807-29816Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). On the basis of its primary structure, UGL was categorized as a member of the glycoside hydrolase family 88 in the CAZy database (19Mori S. Akao S. Nankai H. Hashimoto W. Mikami B. Murata K. Protein Expr. Purif. 2003; 29: 77-84Crossref PubMed Scopus (14) Google Scholar, 20Cantarel B.L. Coutinho P.M. Rancurel C. Bernard T. Lombard V. Henrissat B. Nucleic. Acids Res. 2009; 37: D233-D238Crossref PubMed Scopus (4193) Google Scholar) after we first identified the gene for Bacillus sp. GL1 UGL (BacillusUGL) (21Hashimoto W. Kobayashi E. Nankai H. Sato N. Miya T. Kawai S. Murata K. Arch. Biochem. Biophys. 1999; 368: 367-374Crossref PubMed Scopus (40) Google Scholar). The putative genes for UGL are present in the genome of various pathogenic bacteria, such as streptococci, enterococci, and vibrios. We recently clarified enzymatic characteristics of UGLs from pathogenic streptococci, including Streptococcus agalactiae, Streptococcus pneumoniae, and Streptococcus pyogenes, and determined the crystal structure of S. agalactiae UGL (SagUGL) (22Maruyama Y. Nakamichi Y. Itoh T. Mikami B. Hashimoto W. Murata K. J. Biol. Chem. 2009; 284: 18059-18069Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The enzyme gene is inducibly transcribed in S. agalactiae cells grown in the presence of glycosaminoglycan, indicating that the bacterium produces UGL as well as polysaccharide lyase for complete degradation of glycosaminoglycans. All of the streptococcal UGLs that have been characterized to date actively degrade unsaturated chondroitin and hyaluronan disaccharides and exhibit a preference for sulfated chondroitin disaccharide, especially for Δ6S, an unsaturated chondroitin disaccharide with a sulfate group at the C-6 position of the GalNAc residue. This study deals with the identification of loop movement for substrate binding and structural determinants for substrate specificity in streptococcal UGL through x-ray crystallography of SagUGL in complex with Δ6S, kinetics of site-directed mutants, and molecular conversion of bacterial UGLs with altered substrate specificity. Unsaturated glycosaminoglycan disaccharides were purchased from Seikagaku Biobusiness or Sigma-Aldrich. Silicagel 60/Kieselguhr F254 TLC plates were obtained from Merck. DEAE-Toyopearl 650M was from Tosoh. HiLoad 16/60 Superdex 75pg and Mono Q 10/100 GL were from GE Healthcare. Restriction endonucleases and DNA-modifying enzymes were from Toyobo. Other analytical grade chemicals were obtained from commercial sources. For expression of SagUGL and BacillusUGL, Escherichia coli strain HMS174(DE3) cells transformed with pET21b-SagUGL (22Maruyama Y. Nakamichi Y. Itoh T. Mikami B. Hashimoto W. Murata K. J. Biol. Chem. 2009; 284: 18059-18069Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) and E. coli strain BL21(DE3) cells transformed with pET3a-BacillusUGL (19Mori S. Akao S. Nankai H. Hashimoto W. Mikami B. Murata K. Protein Expr. Purif. 2003; 29: 77-84Crossref PubMed Scopus (14) Google Scholar) were aerobically cultured at 30 °C in LB medium (23Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar) supplemented with sodium ampicillin (0.1 mg/ml). When the turbidity at 600 nm reached 0.3–0.7, isopropyl β-d-thiogalactopyranoside was added to the culture at a final concentration of 0.1 mm, and the cells were further cultured at 16 °C for 44 h. E. coli cells harboring pET21b-SagUGL or pET3a-BacillusUGL were grown in LB medium, collected by centrifugation at 6,700 × g and 4 °C for 5 min, and resuspended in 20 mm potassium phosphate (pH 7.0). The cells were ultrasonically disrupted (Insonator model 201M, Kubota) at 9 kHz and 0 °C for 5 min, and the clear solution obtained by centrifugation at 28,000 × g and 4 °C for 20 min was used as a cell extract. SagUGL and BacillusUGL were purified from the cell extract to homogeneity by several steps of column chromatography (19Mori S. Akao S. Nankai H. Hashimoto W. Mikami B. Murata K. Protein Expr. Purif. 2003; 29: 77-84Crossref PubMed Scopus (14) Google Scholar, 22Maruyama Y. Nakamichi Y. Itoh T. Mikami B. Hashimoto W. Murata K. J. Biol. Chem. 2009; 284: 18059-18069Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Briefly, SagUGL and BacillusUGL were purified by anion exchange chromatography (DEAE-Toyopearl 650M) followed by gel filtration chromatography (HiLoad 16/60 Superdex 75 pg) and finally by anion exchange chromatography (Mono Q 10/100 GL). The degree of purification was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (24Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207522) Google Scholar). The products derived from unsaturated glycosaminoglycan disaccharides through the reaction of bacterial UGLs were separated by TLC using a solvent system of 1-butanol/acetic acid/water (3:2:2, v/v). The products were visualized by heating the TLC plates at 130 °C for 5 min after spraying with 10% (v/v) sulfuric acid in ethanol. Purified SagUGL enzymes of wild type (WT) and mutant D175N with Asp-175 replaced by Asn were concentrated by ultrafiltration using Centriprep (10,000 molecular weight cutoff) (Millipore) to 10 and 5 mg/ml, respectively. Both WT and D175N were crystallized by sitting drop vapor diffusion. The 3 μl of proteins were mixed with an equal volume of a reservoir solution. The reservoir solution for WT crystallization contained 30% (w/v) polyethylene glycol 200, 1% (w/v) polyethylene glycol 3000, and 0.1 m Hepes (pH 7.0). WT crystals grew up at 20 °C for a week. The reservoir solution for D175N crystallization included 40% (w/v) ethylene glycol, 5% (w/v) polyethylene glycol 3000, and 0.1 m Hepes (pH 7.5). D175N was crystallized at 20 °C for a month. To prepare a complex form of SagUGL and Δ6S, the D175N crystal was soaked at 20 °C for 10 min in a reservoir solution containing 0.2 m Δ6S before x-ray diffraction experiments. Crystals were placed in a cold nitrogen gas stream at −173 °C. X-ray diffraction images of crystals were collected using a Jupiter 210 charged-coupled device detector (Rigaku) for the WT crystal or a Quantum 210 charged-coupled device detector (Area Detector Systems Corp.) for D175N crystals with synchrotron radiation at a wavelength of 1.00 Å at the BL-38B1 station of SPring-8 (Hyogo, Japan). The data were processed and scaled with the HKL2000 program (25Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38609) Google Scholar). The structure was determined through molecular replacement with the Molrep program (26Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4174) Google Scholar) supplied in the CCP4 interface program package (27Collaborative Computational Project Acta Crystallogr. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar) by using previously determined coordinates of SagUGL WT (Protein Data Bank code 2ZZR) as an initial model. Structure refinement was conducted with the Refmac5 program (28Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13911) Google Scholar). Randomly selected 5% reflections were excluded from refinement and used to calculate Rfree. After each refinement cycle, the model was adjusted manually using the winCoot program (29Emsley P. Cowtan K. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23605) Google Scholar). Water molecules were incorporated where the difference in density exceeded 3.0 σ above the mean and the 2Fo − Fc map showed a density of more than 1.0 σ. The structure of the enzyme-sugar complex was refined using the Refmac5 and the winCoot program with the chondroitin disaccharide parameter file constructed using PRODRG (50Schuettelkopf A.W. Van Aalten D.M.F. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 1355-1363Crossref PubMed Scopus (4356) Google Scholar). Protein models were superimposed, and their root mean square deviation was determined with the LSQKAB program (30Kabsch W. Acta Crystallogr. A. 1976; 32: 922-923Crossref Scopus (2379) Google Scholar), a part of the CCP4 program package. Final model quality was checked with the PROCHECK program (31Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Figures for protein structures were prepared using the PyMOL program (32DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific LLC, San Carlos, CA2004Google Scholar). Electric charge on the molecular surface of bacterial UGLs was calculated using the APBS program (33Baker N.A. Sept D. Joseph S. Holst M.J. McCammon J.A. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 10037-10041Crossref PubMed Scopus (5903) Google Scholar). Coordinates used in this work were taken from the Protein Data Bank of the Research Collaboratory for Structural Bioinformatics (34Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic. Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (27906) Google Scholar). Thr-235, Ser-365, Ser-368, and Lys-370 of SagUGL were substituted with Ala, His, Gly, and Ile, respectively, and His-210, His-339, Gly-342, and Ile-344 of BacillusUGL were substituted with Arg, Ser, Ser, and Lys, respectively. UGL mutants were constructed using a QuikChange site-directed mutagenesis kit (Stratagene). The plasmid pET21b-SagUGL (22Maruyama Y. Nakamichi Y. Itoh T. Mikami B. Hashimoto W. Murata K. J. Biol. Chem. 2009; 284: 18059-18069Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) or pET3a-BacillusUGL (19Mori S. Akao S. Nankai H. Hashimoto W. Mikami B. Murata K. Protein Expr. Purif. 2003; 29: 77-84Crossref PubMed Scopus (14) Google Scholar) was used as a PCR template, and the synthetic oligonucleotides used as sense and antisense primers are shown in supplemental Table S1. PCR was carried out using KOD-FX polymerase (Toyobo) in place of Pfu polymerase. Mutations were confirmed by the dideoxy chain termination method (35Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U.S.A. 1977; 74: 5463-5467Crossref PubMed Scopus (52766) Google Scholar) using automated DNA sequencer model 3730xl (Applied Biosystems). The cells of the E. coli host strain (HMS174(DE3)) were transformed with the mutant plasmids. Expression and purification of the mutants were conducted by using the same procedures as for SagUGL or BacillusUGL WT as described above. DNA manipulations such as plasmid isolation, subcloning, transformation, and gel electrophoresis were performed as described (23Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Kinetics parameters of SagUGLs (WT, T235A, S365H, S368G, and K370I) and BacillusUGLs (WT, H339S, G342S, and I344K) toward Δ6S or sulfate-free unsaturated chondroitin disaccharide (Δ0S) were determined as follows. The activity of UGLs was assayed at 30 °C by monitoring the decrease in absorbance at 235 nm arising from the double bond (molar extinction coefficient ϵ235 = 4,800 m−1 cm−1) in the substrates. The reaction mixtures consisted of substrate, 20 mm Tris-HCl (pH 7.5), and enzyme. The range of substrate concentration was fixed at 0.05–1.0 mm because the absorbance at 235 nm of the substrate at over 1.0 mm exceeded measurement limitations on the spectrometer. Km and kcat were calculated using the Michaelis-Menten equation with KaleidaGraph software (Synergy Software). In our previous studies (21Hashimoto W. Kobayashi E. Nankai H. Sato N. Miya T. Kawai S. Murata K. Arch. Biochem. Biophys. 1999; 368: 367-374Crossref PubMed Scopus (40) Google Scholar, 22Maruyama Y. Nakamichi Y. Itoh T. Mikami B. Hashimoto W. Murata K. J. Biol. Chem. 2009; 284: 18059-18069Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), Bacillus and streptococcal UGLs have been identified to degrade unsaturated chondroitin and hyaluronan disaccharides. Both chondroitin and hyaluronan include β-GlcUA as a component, whereas α-IdoUA, a C-5 epimer of GlcUA, is predominant (ratio in uronates, >70%) in heparin molecules (3Gandhi N.S. Mancera R.L. Chem. Biol. Drug Des. 2008; 72: 455-482Crossref PubMed Scopus (749) Google Scholar). There is also a difference in glycoside bond pattern between chondroitin/hyaluronan and heparin/heparan sulfate. The 1→3 glycoside bond is present between uronate and amino sugar residues in chondroitin and hyaluronan, although both heparin and heparan sulfate contain the 1→4 glycoside bond between uronate and amino sugar residues. Thus, the enzyme activity of SagUGL was investigated using unsaturated heparin disaccharides with and without sulfate group(s) as a substrate. The sulfate-free unsaturated heparin disaccharide was completely degraded to unsaturated uronic acid and GlcNAc by SagUGL (Fig. 1B, lane 5). In combination with previously reported data, this result indicates that the enzyme acts on unsaturated α-IdoUA (ΔIdoUA) as well as unsaturated β-GlcUA residues in substrates and cleaves both glycoside bonds 1→3 and 1→4. The formation of a double bond between C-4 and C-5 atoms leads to loss of epimerization in GlcUA and IdoUA. In fact, C-3, C-4, C-5, and C-6 atoms of ΔGlcUA were determined to be located in a single plane through structural analysis of UGL and unsaturated glycosaminoglycan disaccharide (18Itoh T. Hashimoto W. Mikami B. Murata K. J. Biol. Chem. 2006; 281: 29807-29816Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Thus, the degradation of both unsaturated chondroitin and heparin disaccharides by UGL was chemically appropriate because each nonreducing terminus showed the same conformation. The activity of UGL on ΔGlcUA- and ΔIdoUA-containing glycosaminoglycan disaccharides suggests that each glycosaminoglycan in mammalian extracellular matrices is first depolymerized to unsaturated disaccharides by a specific polysaccharide lyase, and the resultant unsaturated disaccharides are degraded to the constituent monosaccharides by a single enzyme, UGL. Sulfate-bound disaccharides, such as unsaturated heparin disaccharides with sulfate group(s) at either or both the C-6 position of the GlcNAc residue or the nitrogen position of the GlcN residue, were also degraded by SagUGL (supplemental Fig. S1). Similar to chondroitin and heparin, heparan sulfate and dermatan sulfate include IdoUA and/or GlcUA and sulfate group(s) in their molecules (3Gandhi N.S. Mancera R.L. Chem. Biol. Drug Des. 2008; 72: 455-482Crossref PubMed Scopus (749) Google Scholar). Therefore, streptococcal UGLs are considered as one of the key enzymes for degradation of all uronate-including glycosaminoglycans, chondroitin, hyaluronan, heparin, heparan sulfate, and dermatan sulfate after reactions of polysaccharide lyases. Streptococcal UGLs, including SagUGL, showed a preference for sulfate-bound unsaturated disaccharides from glycosaminoglycans (e.g. chondroitin and heparin) (22Maruyama Y. Nakamichi Y. Itoh T. Mikami B. Hashimoto W. Murata K. J. Biol. Chem. 2009; 284: 18059-18069Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Because this preference of streptococcal UGLs was thought to be dependent on substrate binding, catalytic action, or both, SagUGL and BacillusUGL were kinetically analyzed using sulfate-free (Δ0S) and -bound (Δ6S) unsaturated chondroitin disaccharides as substrates (Table 1). We interpreted the kinetic parameters listed in Table 1 as follows. We treated kinetic parameters with higher Michaelis constants (Km over 1 mm) as estimations because kinetic studies of bacterial UGLs with the higher concentrations (over 1 mm) of substrate were difficult to perform because of their high absorbance at 235 nm. We determined kinetics parameters of SagUGL toward Δ0S and Δ6S. The Km value (1.27 mm) toward Δ0S was over 10-fold higher than that (0.100 mm) toward Δ6S, whereas turnover number (kcat) toward Δ0S was about 3.8-fold smaller than that toward Δ6S. In comparison with the great difference in the affinity for substrate (Km), the difference in kcat was considered to be insignificant. This result indicates that the enzyme exhibits a higher affinity for Δ6S than Δ0S, and its substrate specificity mostly depends on substrate binding. In contrast, BacillusUGL showed a preference for the unsulfated substrate rather than the sulfated substrate because of its high affinity (around 50-fold), although the differences in kcat of the enzyme toward Δ0S and Δ6S were less than 4-fold.TABLE 1Kinetic parameters of bacterial UGLsΔ6SΔ0SKmkcatkcat/KmKmkcatkcat/Kmmms−1s−1 mm−1mms−1s−1 mm−1SagUGLWT0.100 ± 0.028310.2 ± 0.6621021.27 ± 0.09412.69 ± 0.1282.12T235A1.75 ± 0.3443.96 ± 0.5542.26NDaND, kinetic parameters could not be determined due to the very low activity.NDNDS365H2.36 ± 0.1603.85 ± 0.1991.630.763 ± 0.2151.69 ± 0.2552.21S368G0.191 ± 0.026924.8 ± 1.121301.40 ± 0.33210.8 ± 1.717.71K370I1.47 ± 0.19141.6 ± 3.8428.20.371 ± 0.06923.97 ± 0.29710.7BacillusUGLWT18.6 ± 6.6154.9 ± 18.32.950.381 ± 0.039414.1 ± 0.62536.8H339S7.19 ± 1.5424.5 ± 4.703.410.861 ± 0.14716.9 ± 1.4019.6G342S7.00 ± 2.6520.9 ± 7.092.990.504 ± 0.040518.2 ± 0.66536.1I344K2.20 ± 0.10539.8 ± 1.4118.10.566 ± 0.092823.8 ± 1.8642.0a ND, kinetic parameters could not be determined due to the very low activity. Open table in a new tab Based on the kinetics of SagUGL and BacillusUGL, we conclude that the activity on the sulfated substrate was mainly dependent on the affinity for the substrate rather than turnover number. This lesser effect of kcat on substrate specificity was likely due to the multiple actions of UGL strictly on ΔGlcUA but not on amino sugar (18Itoh T. Hashimoto W. Mikami B. Murata K. J. Biol. Chem. 2006; 281: 29807-29816Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), i.e. (i) proton donation to the C-4 atom of ΔGlcUA, (ii) deprotonation of the water molecule, (iii) addition of the water molecule to the C-5 atom of ΔGlcUA, (iv) cleavage of the glycoside bond, and (v) conversion of ΔGlcUA to α-keto acid. Hereafter, substrate specificity was analyzed based on the affinity (Km) for the substrate. The substrate specificity of SagUGL is suggestive of its structural features for specific binding to sulfate groups in the substrate. To clarify structural determinants for sulfate binding, we performed x-ray crystallography of the enzyme-substrate complex. In a previous study, SagUGL was crystallized in a drop solution using ammonium sulfate as the major precipitant, and the crystal structure of the ligand-free enzyme was determined at 1.75-Å resolution (22Maruyama Y. Nakamichi Y. Itoh T. Mikami B. Hashimoto W. Murata K. J. Biol. Chem. 2009; 284: 18059-18069Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). In this study, we conducted many experiments to prepare the enzyme-substrate complex through a soaking treatment of crystals in the substrate solution but failed to obtain the complex. Thus, crystallization conditions were rescreened to accommodate the substrate at the active site of the enzyme. Another crystal of SagUGL WT formed in a drop solution using polyethylene glycol as th" @default.
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• W1990636799 title "Structural Determinants in Streptococcal Unsaturated Glucuronyl Hydrolase for Recognition of Glycosaminoglycan Sulfate Groups" @default.
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