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• W2051287611 abstract "Hyaluronate lyase enzymes degrade hyaluronan, the main polysaccharide component of the host connective tissues, predominantly into unsaturated disaccharide units, thereby destroying the normal connective tissue structure and exposing the tissue cells to various endo- and exogenous factors, including bacterial toxins. The crystal structures of Streptococcus pneumoniaehyaluronate lyase with tetra- and hexasaccharide hyaluronan substrates bound in the active site were determined at 1.52- and 2.0-Å resolution, respectively. Hexasaccharide is the longest substrate segment that binds entirely within the active site of these enzymes. The enzyme residues responsible for substrate binding, positioning, catalysis, and product release were thereby identified and their specific roles characterized. The involvement of three residues in catalysis, Asn349, His399, and Tyr408, is confirmed, and the details of proton acceptance and donation within the catalytic machinery are described. The mechanism of processivity of the enzyme is analyzed. The flexibility (allosteric) behavior of the enzyme may be understood in terms of the results of flexibility analysis of this protein, which identified two modes of motion that are also proposed to be involved in the hyaluronan degradation process. The first motion describes an opening and closing of the catalytic cleft located between the α- and β-domains. The second motion demonstrates the mobility of a binding cleft, which may facilitate the binding of the negatively charged hyaluronan to the enzyme. Hyaluronate lyase enzymes degrade hyaluronan, the main polysaccharide component of the host connective tissues, predominantly into unsaturated disaccharide units, thereby destroying the normal connective tissue structure and exposing the tissue cells to various endo- and exogenous factors, including bacterial toxins. The crystal structures of Streptococcus pneumoniaehyaluronate lyase with tetra- and hexasaccharide hyaluronan substrates bound in the active site were determined at 1.52- and 2.0-Å resolution, respectively. Hexasaccharide is the longest substrate segment that binds entirely within the active site of these enzymes. The enzyme residues responsible for substrate binding, positioning, catalysis, and product release were thereby identified and their specific roles characterized. The involvement of three residues in catalysis, Asn349, His399, and Tyr408, is confirmed, and the details of proton acceptance and donation within the catalytic machinery are described. The mechanism of processivity of the enzyme is analyzed. The flexibility (allosteric) behavior of the enzyme may be understood in terms of the results of flexibility analysis of this protein, which identified two modes of motion that are also proposed to be involved in the hyaluronan degradation process. The first motion describes an opening and closing of the catalytic cleft located between the α- and β-domains. The second motion demonstrates the mobility of a binding cleft, which may facilitate the binding of the negatively charged hyaluronan to the enzyme. S. pneumoniae hyaluronate lyase essential dynamics molecular dynamics hyaluronan tetrasaccharide of HA hexasaccharide of HA and HA2, HA3, positions of hyaluronan building blocks of disaccharide for HA numbered from the reducing toward the non-reducing end proton acceptance and donation Streptococcus pneumoniae colonizes predominantly the upper respiratory tract of humans and is a major human pathogenic bacterium. It is one of the key causes of life-threatening disease such as pneumonia, bacteremia, and meningitis (1Mufson M.A. Mandell G.L. Douglas R.G., Jr. Bennett J.E. Principles and Practice of Infectious Diseases. Churchill Levingstone, New York1990Google Scholar). It also causes less threatening diseases that are, however, very prevalent like otitis media and sinusitis (2Stool S.E. Field M.J. Pediatr. Infect. Dis. J. 1989; 8: S11-S14Crossref PubMed Scopus (166) Google Scholar). Pneumococci interact with the host and its tissues through the surface sugars (capsule) and a variety of usually surface-exposed protein molecules. These interactions are essential for the full pathogenicity of these bacteria and are likely involved in the disease-causing processes. The proteins known to be involved in this interaction include, among others, hyaluronate lyase (3Berry A.M. Lock R.A. Thomas S.M. Rajan D.P. Hansman D. Paton J.C. Infect. Immun. 1994; 62: 1101-1108Crossref PubMed Google Scholar, 4Lock R.A. Paton J.C. Hansman D. Microb. Pathog. 1988; 5: 461-467Crossref PubMed Scopus (92) Google Scholar), pneumolysin (5Paton J.C. Berry A.M. Lock R.A. Hansman D. Manning P.A. Infect. Immun. 1986; 54: 50-55Crossref PubMed Google Scholar), pneumococcal surface protein A (6McDaniel L.S. Sheffield J.S. DeLucchi P. Briles D.E. Infect. Immun. 1991; 59: 222-228Crossref PubMed Google Scholar), and pneumococcal surface antigen A (7Jedrzejas M.J. Microbiol. Mol. Biol. Rev. 2001; 65: 187-207Crossref PubMed Scopus (374) Google Scholar, 8Berry A.M. Paton J.C. Infect. Immun. 1996; 64: 5255-5262Crossref PubMed Google Scholar). S. pneumoniae hyaluronate lyase (SpnHL)1 primarily degrades hyaluronan (HA), the predominant polysaccharide component of animal and human connective tissues and the nervous system, into unsaturated disaccharide units as the end products (9Li S. Kelly S.J. Lamani E. Ferraroni M. Jedrzejas M.J. EMBO J. 2000; 19: 1228-1240Crossref PubMed Scopus (149) Google Scholar). Cells in the connective tissues are embedded in the strikingly viscoelastic HA matrix. Hyaluronan is a polymeric glycan composed of linear repeats of a few hundred to as many as 20,000 or more disaccharide units of glucuronic acid and N-acetylglucosamine. The glycosidic linkage present within the disaccharide unit is β-1,3, whereas the disaccharide units are connected with the β-1,4-glycosidic linkage. The HA metabolism seems to be more significant than the metabolism of other polysaccharides in vivo due to the large turnover of this sugar. In human, one-third of the HA (about 5 g) is turned over daily (10McCourt P.A.G. Matrix Biol. 1999; 18: 427-432Crossref PubMed Scopus (29) Google Scholar). The rapid turnover rate facilitates the use of HA and its degradation products in many physiological processes, such as cell differentiation and development (11Toole B.P. Goldberg R.L. Chi-Rosso G. Underhill C.B. Orkin R.W. Trelstad R.C. The Role of Extracellular Matrix in Development. Alan R. Liss, Inc., New York1984Google Scholar), cell proliferation, recognition, locomotion, and the immunological responses (12Laurent T.C. Fraser R.E. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2078) Google Scholar). Enzymes of either mammalian (including host or human enzymes) or bacterial origin degrade HA at the β-1,4-linkage exerting the eno- and exogenous pressures on the host. Bacteria secrete hyaluronate lyases to degrade HA, and these enzymes usually produce unsaturated di-, tetra- (HA4), or hexasaccharides (HA6). The mechanism of this degradation by Streptococcus species hyaluronate lyase, which produces primarily unsaturated disaccharides of HA, 2-acetamido-2-deoxy-3-O-(β-d-gluco-4-enepyranosyluronic acid)-d-glucose, as the end products, was recently proposed and involves the elimination reaction introducing an unsaturated bond to the product (9Li S. Kelly S.J. Lamani E. Ferraroni M. Jedrzejas M.J. EMBO J. 2000; 19: 1228-1240Crossref PubMed Scopus (149) Google Scholar). Mammals, on the other hand, express hyaluronidases to degrade HA and to produce relatively longer oligosaccharides (13Jedrzejas M.J. Crit. Rev. Biochem. Mol. Biol. 2000; 35: 221-251Crossref PubMed Scopus (72) Google Scholar). The detailed atomic mechanism of HA degradation by mammal hyaluronidases is still largely unknown due, in part, to the lack of structural information. This process likely relies on the displacement mechanism based on hydrolysis, similar to that of cellulases, to degrade the HA substrate (13Jedrzejas M.J. Crit. Rev. Biochem. Mol. Biol. 2000; 35: 221-251Crossref PubMed Scopus (72) Google Scholar). Mammalian hyaluronidases (mainly bovine hyaluronidase) are, however, widely used in clinics, for example as an additive to the local anesthesia for the fast spreading and penetration of medications through tissues (14Courtiss E.H. Ransil B.J. Russo J. Plast. Reconstr. Surg. 1995; 95: 876-883Crossref PubMed Google Scholar, 15Menzel E.J. Farr C. Cancer Lett. 1998; 131: 3-11Crossref PubMed Scopus (280) Google Scholar). The structure of the native SpnHL (9Li S. Kelly S.J. Lamani E. Ferraroni M. Jedrzejas M.J. EMBO J. 2000; 19: 1228-1240Crossref PubMed Scopus (149) Google Scholar) and its complex with the disaccharide products of HA degradation (16Ponnuraj K. Jedrzejas M.J. J. Mol. Biol. 2000; 299: 885-895Crossref PubMed Scopus (88) Google Scholar) were recently reported. This structural information together with further characterization of the enzyme (17Kelly S.J. Taylor K.B., Li, S. Jedrzejas M.J. Glycobiology. 2001; 11: 297-304Crossref PubMed Scopus (45) Google Scholar, 18Li S. Taylor K.B. Kelly S.J. Jedrzejas M.J. J. Biol. Chem. 2001; 276: 15125-15130Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) allowed for the formulation of the proposed mechanism of HA degradation (9Li S. Kelly S.J. Lamani E. Ferraroni M. Jedrzejas M.J. EMBO J. 2000; 19: 1228-1240Crossref PubMed Scopus (149) Google Scholar, 13Jedrzejas M.J. Crit. Rev. Biochem. Mol. Biol. 2000; 35: 221-251Crossref PubMed Scopus (72) Google Scholar). Briefly, the mechanism of catalysis, termed proton acceptance and donation, involves a five-step process involving neutralization of the carboxylate of a glucuronic moiety, extraction of a proton from C-5 followed by a double bond formation in the glucuronate and breaking of the β-1,4-glycosidic linkage. Additional comparison to other enzymes degrading glycans likeStreptococcus agalactiae hyaluronate lyase (19Li S. Jedrzejas M.J. J. Biol. Chem. 2001; 276: 41407-41416Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar),Flavobacterium heparinum chondroitin AC lyase (20Huang W. Boju L. Tkalec L., Su, H. Yang H.O. Gunay N.S. Linhardt R.J. Kim Y.S. Matte A. Cygler M. Biochemistry. 2001; 40: 2359-2372Crossref PubMed Scopus (77) Google Scholar), andSphingomonas species alginate lyase A1-III (21Yoon H.J. Hashimoto W. Miyake O. Murata K. Mikami B. J. Mol. Biol. 2001; 307: 9-16Crossref PubMed Scopus (78) Google Scholar) allowed for generalization of the pneumococcal enzyme mechanism and suggested a similar mechanism of action for these additional enzymes (13Jedrzejas M.J. Crit. Rev. Biochem. Mol. Biol. 2000; 35: 221-251Crossref PubMed Scopus (72) Google Scholar). Here we report the structures of the S. pneumoniae hyaluronate lyase complexes with its substrates, the tetra- and hexasaccharide units of hyaluronan, and we discuss their implications for the mechanism of action of the enzyme. The SpnHL Y408F mutant cloning, overexpression in Escherichia coli (pMJJ004), and purification were reported previously (9Li S. Kelly S.J. Lamani E. Ferraroni M. Jedrzejas M.J. EMBO J. 2000; 19: 1228-1240Crossref PubMed Scopus (149) Google Scholar, 22Jedrzejas M.J. Mewbourne R.B. Chantalat L. McPherson D.T. Protein Expression Purif. 1998; 13: 83-89Crossref PubMed Scopus (47) Google Scholar). The mutant enzyme was concentrated to 5 mg/ml in 10 mm Tris-HCl, pH 7.4, 2 mm EDTA, and 1 mmdl-dithiothreitol and stored at −80 °C until their use. The tetra- and hexasaccharide substrates were also obtained as described previously (17Kelly S.J. Taylor K.B., Li, S. Jedrzejas M.J. Glycobiology. 2001; 11: 297-304Crossref PubMed Scopus (45) Google Scholar, 18Li S. Taylor K.B. Kelly S.J. Jedrzejas M.J. J. Biol. Chem. 2001; 276: 15125-15130Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). To prevent degradation they were stored frozen at −80 °C in 10 mm Tris-HCl buffer, pH 8.0, until their use. The crystals of Y408F mutant version of SpnHL were obtained using the same conditions as for the native enzyme crystals (100 mmsodium cacodylate buffer, pH 6.0, 2.9 m ammonium sulfate) but with the addition of 5 mm EDTA (23Jedrzejas M.J. Chantalat L. Mewbourne R.B. J. Struct. Biol. 1998; 121: 73-75Crossref PubMed Scopus (26) Google Scholar). Crystallization of Y408F mutant enzyme was accomplished using the vapor diffusion hanging drop method in 24-well Linbro culture plates using equal volumes of the protein sample and the reservoir solution (2 μl of each) at 22 °C (24-Google Scholar, 25McPherson A. Crystallization of Biological Molecules. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York1999Google Scholar). The protein and reservoir solution were mixed and equilibrated against 1.0 ml of reservoir solution. Diffraction quality rectangular block shaped crystals of Y408F (0.5 × 0.3 × 0.3 mm) were grown within several days. The crystals of Y408F SpnHL were then soaked at room temperature with 20 mm tetra- and hexasaccharide substrates (both in 10 mm Tris-HCl buffer at pH 8.0) for 10 h directly before the x-ray diffraction data collection. The crystal soaking solution contained the crystallization conditions (100 mm sodium cacodylate buffer, pH 6.0) with increased concentration of the precipitation agent, ammonium sulfate, to 3.2 m. The crystal was cryo-protected using the same conditions as for the native crystals (23Jedrzejas M.J. Chantalat L. Mewbourne R.B. J. Struct. Biol. 1998; 121: 73-75Crossref PubMed Scopus (26) Google Scholar) and flash freezing them at −170 °C in a nitrogen flow using a Cryostream Cooler (Cryosystems, Oxford, UK). The diffraction data sets were collected using synchrotron radiation at the wavelength of 1.0 Å at the 19-BM beamline at the Argonne National Laboratory, Advanced Photon Source, Structural Biology Center using an Oxford 3 × 3 charged-coupled device detector. The diffraction limits of the crystals were 1.52- and 2.0-Å resolution for the tetra- and hexasaccharide complexes, respectively. The data were processed and scaled using the HKL2000 package (26Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38572) Google Scholar). The crystals of complexes were isomorphous to the native ones. The final data processing parameters are reported in Table I.Table ICrystallographic statistics of diffraction data and structure refinement of S. pneumoniae hyaluronate lyase complexes with tetra- and hexasaccharide hyaluronan substratesSpnHL-HA4complexSpnHL-HA6 complexSpace groupP212121P212121Unit cell dimensions (Å) a = 83.7, b = 103.8, c = 101.3 a = 83.7, b = 103.6, c = 101.3Resolution range (Å)50.00–1.5250.00–2.00Measured reflections904,361407,437Unique reflections124,92760,259Completeness (last shell) (%)1-aThe last shell is defined as 1.52–1.59 Å for the tetrasaccharide complex and 2.0–2.07 Å for the hexasaccharide complex.93.1 (49.8)99.9 (99.4)Rmerge1-bRmerge=Σ‖Ii−'I'/ΣIi×100 whereI i is the intensity of an individual reflection, and 'I' is the mean of all reflection.(last shell) (%)8.0 (61.1)16.7 (91.0)Final model719 amino acids, 585 waters721 amino acids, 590 watersRcryst1-cRcrys = Σ∥F p ‖ − ‖F calc∥/Σ‖F p ‖ × 100 where F p and F calc are the measured and the calculated structure factors, respectively. (last shell)19.00 (33.0)22.4 (32.4)Rfree1-dRfree is as defined by Brunger and Krukowski (55). (last shell)23.9 (39.5)24.3 (38.4)R.m.s.d.1-eR.m.s.d., root mean square deviation. bonds (Å)0.0060.007R.m.s.d.1-eR.m.s.d., root mean square deviation. angles1.2351.305Average B-factors (Å2)Whole protein45.0826.24α-Domain48.4029.18β-Domain41.3623.20Water molecules53.4834.22Substrates67.2720.211-a The last shell is defined as 1.52–1.59 Å for the tetrasaccharide complex and 2.0–2.07 Å for the hexasaccharide complex.1-b Rmerge=Σ‖Ii−'I'/ΣIi×100 whereI i is the intensity of an individual reflection, and 'I' is the mean of all reflection.1-c Rcrys = Σ∥F p ‖ − ‖F calc∥/Σ‖F p ‖ × 100 where F p and F calc are the measured and the calculated structure factors, respectively.1-d Rfree is as defined by Brunger and Krukowski (55Brunger A.T. Krukowski A. Acta Crystallogr. Sect. A. 1990; 46: 585-593Crossref PubMed Scopus (600) Google Scholar).1-e R.m.s.d., root mean square deviation. Open table in a new tab The native SpnHL crystal structure without water molecules (9Li S. Kelly S.J. Lamani E. Ferraroni M. Jedrzejas M.J. EMBO J. 2000; 19: 1228-1240Crossref PubMed Scopus (149) Google Scholar) was used as the model for the complex structure solution. The Rfree flag was assigned to 1 (1,249 reflections) and 2% (1,205 reflections) reflections for the tetra- and hexasaccharide complexes, respectively, to validate the refinement progress (27Brunger A.T. Nature. 1992; 355: 472-474Crossref PubMed Scopus (3864) Google Scholar). Initially, a round of rigid body refinements using only the model was performed using X-PLOR (28Brunger A.T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16967) Google Scholar). The Tyr408 was manually mutated to Phe using the O program (29Jones T.A. Zhou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13011) Google Scholar). Refinements progress using program X-PLOR with the positional and simulated annealing protocols (28Brunger A.T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16967) Google Scholar, 30Rice L.M. Brunger A.T. Proteins Struct. Funct. Genet. 1994; 19: 277-290Crossref PubMed Scopus (382) Google Scholar) and was traced using inspection on graphics with program O (29Jones T.A. Zhou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13011) Google Scholar). The structures were refined against 124,927 and 60,437 reflections for the tetra- and hexasaccharide complexes, respectively, and utilizing all data from 50 Å to the highest resolution available and a 2ς(F) cut-off (Table I). The electron density for the substrates was then clearly identified, and the incorporation of the substrates into this density was followed. The topologies and parameter files for both substrates were manually created following our earlier studies with disaccharide substrates and carefully corrected to reflect ideal stereochemical values (16Ponnuraj K. Jedrzejas M.J. J. Mol. Biol. 2000; 299: 885-895Crossref PubMed Scopus (88) Google Scholar). Additional refinements including individual restrained B-factor refinements, inspection, and manipulation of structure on graphics using O together with incorporation of water molecules placed into 3ς peaks in ς-A weighted F o − F c maps following standard criteria were performed. After refinement, water molecules whose positions were not supported by electron density, at 1ς contouring, in ς-A weighted 2F o −F c maps were deleted. A variety of stereochemical (31Kleywegt G.J. Jones T.A. Acta Crystallogr. Sect. D Biol. Crystallogr. 1996; 52: 829-832Crossref PubMed Scopus (164) Google Scholar) and other analyses (29Jones T.A. Zhou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13011) Google Scholar, 32Laskowski R.A. MacArthur M.W. Moss D.S. Thorton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) was periodically performed in order to locate possible model errors. The tetra- and hexasaccharide substrate molecules and 685 and 590 waters were incorporated, respectively, into the final structures of these two complexes. Final refinement parameters are reported in Table I. The CONCOORD program (33de Groot B.L. van Aalten D.M. Scheek R.M. Amadei A. Vriend G. Berendsen H.J. Proteins Struct. Funct. Genet. 1997; 29: 240-251Crossref PubMed Scopus (227) Google Scholar, 34de Groot B.L. Vriend G. Berendsen H.J. J. Mol. Biol. 1999; 286: 1241-1249Crossref PubMed Scopus (61) Google Scholar) generates uncorrelated protein structures fulfilling a set of upper and lower interatomic distance limits. These limits are derived from experimental structures through measurement of interatomic distances and prediction of the interaction strength of the involved atoms. Thus, the separation of strongly interacting atoms was allowed to vary only slightly from the observed value, whereas weaker interactions have more relaxed limits. Special consideration was given to interacting atoms within the same secondary structure element in order to ensure the preservation of secondary structure. Although detailed aspects of dynamic variation may not be reproduced by this method, good qualitative agreement has been found between the results of the conventional molecular dynamics and CONCOORD simulations (33de Groot B.L. van Aalten D.M. Scheek R.M. Amadei A. Vriend G. Berendsen H.J. Proteins Struct. Funct. Genet. 1997; 29: 240-251Crossref PubMed Scopus (227) Google Scholar, 34de Groot B.L. Vriend G. Berendsen H.J. J. Mol. Biol. 1999; 286: 1241-1249Crossref PubMed Scopus (61) Google Scholar, 35Kleinjung J. Bayley P. Fraternali F. FEBS Lett. 2000; 470: 257-262Crossref PubMed Scopus (17) Google Scholar). Trajectories containing 500 conformations were calculated. The essential dynamics (ED) was performed by Gromacs 3.0 (36Berendsen H.J.C. van der Spoel D. van Drunen R. Comp. Phys. Comm. 1995; 91: 43-56Crossref Scopus (7260) Google Scholar, 37Lindahl E. Hess B. van der Spoel D. J. Mol. Model. 2001; 7: 306-317Crossref Google Scholar). This method is based on the diagonalization of the covariance matrix of atomic fluctuations, which yields a set of eigenvalues and eigenvectors (38Amadei A. Linssen A.B. Berendsen H.J. Proteins Struct. Funct. Genet. 1993; 17: 412-425Crossref PubMed Scopus (2636) Google Scholar). The eigenvectors indicate directions in a 3N dimensional space (whereN = number of atoms) and describe concerted fluctuations of the atoms, constructed such that the amount of fluctuation along a small number of eigenvectors is maximized. The eigenvalues reflect the magnitude of the fluctuation along with the respective eigenvectors. The central hypothesis of this method is that the motions along the eigenvectors with the largest eigenvalues are essential for describing the function. The motion along any desired eigenvector can be inspected by projecting the frames from the trajectory onto that eigenvector. The figures were prepared using Ribbons (39Carson M. Methods Enzymol. 1997; 277: 493-505Crossref PubMed Scopus (656) Google Scholar), Grasp (40Nicholls A. Sharp K.A. Honig B. Proteins Struct. Funct. Genet. 1991; 11: 281-296Crossref PubMed Scopus (5316) Google Scholar), O (29Jones T.A. Zhou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13011) Google Scholar), and Molscript (41Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar). The nativeS. pneumoniae hyaluronate lyase enzyme used in our studies was active in the crystallized form. When crystal was combined with the hyaluronan substrates, the degradation to disaccharides was observed. This led to three-dimensional structures for the complexes identical to that of the complex with the disaccharide product reported earlier (16Ponnuraj K. Jedrzejas M.J. J. Mol. Biol. 2000; 299: 885-895Crossref PubMed Scopus (88) Google Scholar) (data not shown). The presence in crystals of EDTA, to chelate divalent cations such as calcium, which are required for full enzyme activity, was not sufficient to totally prevent substrate degradation by the enzyme (data not shown). The use of an inactive mutant of the enzyme was, therefore, necessary to obtain complexes of the hyaluronan-derived substrates larger than disaccharide HA with the enzyme. Our earlier studies (9Li S. Kelly S.J. Lamani E. Ferraroni M. Jedrzejas M.J. EMBO J. 2000; 19: 1228-1240Crossref PubMed Scopus (149) Google Scholar) showed that the Y408F mutant of the enzyme is essentially inactive, and for this reason this mutant form of the enzyme was chosen for our studies. Indeed, the tetra- and hexasaccharide HA substrates were not degraded by the enzyme during complex formation in the crystals. The Y408F mutant enzyme consisted of residues Ala168 to Gln892 of the full-length protein and a C-terminal His6 tag engineered for the ease of purification (22Jedrzejas M.J. Mewbourne R.B. Chantalat L. McPherson D.T. Protein Expression Purif. 1998; 13: 83-89Crossref PubMed Scopus (47) Google Scholar). The tetra- and hexasaccharide hyaluronan substrates were chosen because our earlier modeling studies (9Li S. Kelly S.J. Lamani E. Ferraroni M. Jedrzejas M.J. EMBO J. 2000; 19: 1228-1240Crossref PubMed Scopus (149) Google Scholar, 16Ponnuraj K. Jedrzejas M.J. J. Mol. Biol. 2000; 299: 885-895Crossref PubMed Scopus (88) Google Scholar) suggested that they should fit entirely within the catalytic cleft of the enzyme and not interfere with the crystallization process. These truncated hyaluronan substrates were produced in milligram quantities from a human umbilical cord hyaluronan by a controlled degradation with Streptomyces hyaluroliticus hyaluronate lyase as reported previously (9Li S. Kelly S.J. Lamani E. Ferraroni M. Jedrzejas M.J. EMBO J. 2000; 19: 1228-1240Crossref PubMed Scopus (149) Google Scholar, 42Park Y. Cho S. Linhardt R.J. Biochim. Biophys. Acta. 1997; 1337: 217-226Crossref PubMed Scopus (56) Google Scholar,43Shimada E. Matsumura G. J. Biochem. (Tokyo). 1980; 88: 1015-1023Crossref PubMed Scopus (46) Google Scholar). This enzyme is an eliminase and produces tetra- and hexasaccharides with an unsaturated uronic acid at their non-reducing end as the final degradation products. These products were identified using ion spray mass spectrometry experiments. The crystals of the inactive Y408F mutant form of the enzyme were obtained using the hanging drop vapor diffusion method and essentially the original crystallization condition of the native enzyme reported previously (23Jedrzejas M.J. Chantalat L. Mewbourne R.B. J. Struct. Biol. 1998; 121: 73-75Crossref PubMed Scopus (26) Google Scholar). The crystals of the complexes were obtained by soaking the Y408F mutant enzyme crystals with 20 mm tetra- and hexasaccharides of HA for a relatively short time directly before the freezing the crystals and/or diffraction data collection. The best soaking time was determined to be 10 h. The diffraction data were collected and processed, and structures were solved and refined as described under “Experimental Procedures.” The data in TableI show well refined structures with excellent stereochemistry. In a Ramachandran plot (31Kleywegt G.J. Jones T.A. Acta Crystallogr. Sect. D Biol. Crystallogr. 1996; 52: 829-832Crossref PubMed Scopus (164) Google Scholar), a majority of the residues for the tetra- and hexasaccharide complexes are located in the most favored areas and none in the disallowed or generously allowed areas. These crystal structures were solved at 1.52- and 2.0-Å resolution, respectively (Fig. 1). The tetrasaccharide complex structure contains residues Lys171to Lys889, and three N-terminal residues and three C-terminal residues together with the C-terminal His6 tag were not seen in the electron density. For the hexasaccharide complex one additional residue on each end of the molecule was partially located in the electron density maps and was modeled as alanine. The electron density was very clear for the entire structure of both complexes, including the substrate molecules, with the exception of the very N and C termini of the enzyme where disorder was evident (Fig.2). The structure of the complex with the tetrasaccharide contained 685 ordered water molecules, whereas that of hexasaccharide includes 590 water molecules in addition to protein and substrate molecules. For both substrates, there was only one substrate molecule bound per molecule of the enzyme.Figure 2Electron density maps for the substrate molecules in the structure of their complexes with the enzyme. The figure shows a stereo view of the 2F o −F c electron density maps contoured at 1ς level.a, electron density for tetrasaccharide hyaluronan.b, electron density for hexasaccharide hyaluronan.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The protein components of both crystal structures of the complexes are nearly identical to one another as well as to the structure of the native enzyme (9Li S. Kelly S.J. Lamani E. Ferraroni M. Jedrzejas M.J. EMBO J. 2000; 19: 1228-1240Crossref PubMed Scopus (149) Google Scholar). Briefly, the protein molecules are built from two structural domains connected only by one short and flexible linker peptide consisting from 11 amino acid residues (Fig. 1). The structural domains are the N-terminal α-helical domain (α-domain) and the C-terminal β-sheet domain (β-domain). The α-domain is composed of 13 helices arranged in a α5/α5-barrel structure (Fig. 1). The larger end of this barrel is shaped as a deep and elongated cleft, which faces the β-domain. The β-domain is primarily composed of five anti-parallel β-sheet structures forming a β-sandwich. A part of this domain is in close proximity to the cleft present in the α-domain. However, its contribution to the shape of the cleft is minimal and restricted to two loops between the β-sheets of the β-domain, which contribute to the shape of a part of one edge of the cleft (Fig. 1). The cleft contains the substrate-binding site and catalytic residues. The detailed description of the native structure has been reported elsewhere (9Li S. Kelly S.J. Lamani E. Ferraroni M. Jedrzejas M.J. EMBO J. 2000; 19: 1228-1240Crossref PubMed Scopus (149) Google Scholar). The structures of both tetra- and hexasaccharide HA" @default.
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• W2051287611 date "2002-08-01" @default.
• W2051287611 modified "2023-09-30" @default.
• W2051287611 title "Mechanism of Hyaluronan Degradation byStreptococcus pneumoniae Hyaluronate Lyase" @default.
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