The Mechanisms by Which Family 10 Glycoside Hydrolases Bind Decorated Substrates
2004; Elsevier BV; Volume: 279; Issue: 10 Linguagem: Inglês
10.1074/jbc.m312278200
ISSN1083-351X
AutoresG. Pell, Edward J. Taylor, T.M. Gloster, J.P. Turkenburg, C.M.G.A. Fontes, L.M.A. Ferreira, Tibor Nagy, S. Clark, G.J. Davies, Harry J. Gilbert,
Tópico(s)Enzyme Production and Characterization
ResumoEndo-β-1,4-xylanases (xylanases), which cleave β-1,4 glycosidic bonds in the xylan backbone, are important components of the repertoire of enzymes that catalyze plant cell wall degradation. The mechanism by which these enzymes are able to hydrolyze a range of decorated xylans remains unclear. Here we reveal the three-dimensional structure, determined by x-ray crystallography, and the catalytic properties of the Cellvibrio mixtus enzyme Xyn10B (CmXyn10B), the most active GH10 xylanase described to date. The crystal structure of the enzyme in complex with xylopentaose reveals that at the +1 subsite the xylose moiety is sandwiched between hydrophobic residues, which is likely to mediate tighter binding than in other GH10 xylanases. The crystal structure of the xylanase in complex with a range of decorated xylooligosaccharides reveals how this enzyme is able to hydrolyze substituted xylan. Solvent exposure of the O-2 groups of xylose at the +4, +3, +1, and -3 subsites may allow accommodation of the α-1,2-linked 4-O-methyl-d-glucuronic acid side chain in glucuronoxylan at these locations. Furthermore, the uronic acid makes hydrogen bonds and hydrophobic interactions with the enzyme at the +1 subsite, indicating that the sugar decorations in glucuronoxylan are targeted to this proximal aglycone binding site. Accommodation of 3′-linked l-arabinofuranoside decorations is observed in the -2 subsite and could, most likely, be tolerated when bound to xylosides in -3 and +4. A notable feature of the binding mode of decorated substrates is the way in which the subsite specificities are tailored both to prevent the formation of "dead-end" reaction products and to facilitate synergy with the xylan degradation-accessory enzymes such as α-glucuronidase. The data described in this report and in the accompanying paper (Fujimoto, Z., Kaneko, S., Kuno, A., Kobayashi, H., Kusakabe, I., and Mizuno, H. (2004) J. Biol. Chem. 279, 9606-9614) indicate that the complementarity in the binding of decorated substrates between the glycone and aglycone regions appears to be a conserved feature of GH10 xylanases. Endo-β-1,4-xylanases (xylanases), which cleave β-1,4 glycosidic bonds in the xylan backbone, are important components of the repertoire of enzymes that catalyze plant cell wall degradation. The mechanism by which these enzymes are able to hydrolyze a range of decorated xylans remains unclear. Here we reveal the three-dimensional structure, determined by x-ray crystallography, and the catalytic properties of the Cellvibrio mixtus enzyme Xyn10B (CmXyn10B), the most active GH10 xylanase described to date. The crystal structure of the enzyme in complex with xylopentaose reveals that at the +1 subsite the xylose moiety is sandwiched between hydrophobic residues, which is likely to mediate tighter binding than in other GH10 xylanases. The crystal structure of the xylanase in complex with a range of decorated xylooligosaccharides reveals how this enzyme is able to hydrolyze substituted xylan. Solvent exposure of the O-2 groups of xylose at the +4, +3, +1, and -3 subsites may allow accommodation of the α-1,2-linked 4-O-methyl-d-glucuronic acid side chain in glucuronoxylan at these locations. Furthermore, the uronic acid makes hydrogen bonds and hydrophobic interactions with the enzyme at the +1 subsite, indicating that the sugar decorations in glucuronoxylan are targeted to this proximal aglycone binding site. Accommodation of 3′-linked l-arabinofuranoside decorations is observed in the -2 subsite and could, most likely, be tolerated when bound to xylosides in -3 and +4. A notable feature of the binding mode of decorated substrates is the way in which the subsite specificities are tailored both to prevent the formation of "dead-end" reaction products and to facilitate synergy with the xylan degradation-accessory enzymes such as α-glucuronidase. The data described in this report and in the accompanying paper (Fujimoto, Z., Kaneko, S., Kuno, A., Kobayashi, H., Kusakabe, I., and Mizuno, H. (2004) J. Biol. Chem. 279, 9606-9614) indicate that the complementarity in the binding of decorated substrates between the glycone and aglycone regions appears to be a conserved feature of GH10 xylanases. The microbial hydrolysis of the plant cell wall into its constituent sugars is one of the major mechanisms by which organic carbon is utilized in the biosphere. The enzymes that catalyze this process are thus of considerable biological and industrial importance. Endo-β-1,4-xylanases (xylanases), which are one of the key components of the repertoire of enzymes that catalyze plant cell wall degradation, hydrolyze the β-1,4 glycosidic bonds linking the xyloside units that comprise the backbone of the polysaccharide xylan (1Brett C.T. Waldren K. Black M. Charlewood B. Physiology and Biochemistry of Plant Cell Walls. Topics in Plant Functional Biology. Vol. 1. Chapman and Hall, London1996Google Scholar). Furthermore, the O-2 and/or O-3 of xylosides within the xylan backbone may be decorated with acetyl and arabinofuranosyl units, whereas 4-O-methyl-d-glucuronic acid (4-O-MeGlcA) 1The abbreviations used are: 4-O-MeGlcA, 4-O-methyl-d-glucuronic acid; GH, glycoside hydrolase family; AX2, arabinoxylobiose; AX3, arabinoxylotriose; OX, oat spelt xylan; RAX, rye arabinoxylan; MX3, aldotetraouronic acid; Xyn10, xylanase from GH10; HPLC, high pressure liquid chromatography; PEG, polyethylene glycol. is exclusively α-1,2-linked (1Brett C.T. Waldren K. Black M. Charlewood B. Physiology and Biochemistry of Plant Cell Walls. Topics in Plant Functional Biology. Vol. 1. Chapman and Hall, London1996Google Scholar). The extent and nature of such decoration varies between plant species. For complete degradation the arabinofuranosyl and acetyl side chains are removed from the polysaccharide by arabinofuranosidases and acetyl xylan esterases, respectively (1Brett C.T. Waldren K. Black M. Charlewood B. Physiology and Biochemistry of Plant Cell Walls. Topics in Plant Functional Biology. Vol. 1. Chapman and Hall, London1996Google Scholar), whereas the α-glucuronidases, which release 4-O-MeGlcA, act only on xylooligosaccharides in which the xylose at the nonreducing end is decorated with the uronic acid (2Nurizzo D. Nagy T. Gilbert H.J. Davies G.J. Structure. 2002; 10: 547-556Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 3Nagy T. Nurizzo D. Davies G.J. Biely P. Lakey J.H. Bolam D.N. Gilbert H.J. J. Biol. Chem. 2003; 278: 20286-20292Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Xylanases, which are generally located in glycoside hydrolase families (GH) 10 and 11 (CAZy website 2CAZy website: afmb.cnrs-mrs.fr/CAZY/. and Ref. 4Henrissat B. Biochem. J. 1991; 280: 309-316Crossref PubMed Scopus (2651) Google Scholar), hydrolyze glycosidic bonds by acid base-assisted catalysis via a double displacement mechanism leading to retention of anomeric configuration at the site of cleavage (5Davies G. Henrissat B. Structure. 1995; 3: 853-859Abstract Full Text Full Text PDF PubMed Scopus (1628) Google Scholar). The crystal structures of xylanases show that GH10 enzymes fold into a (β/α)8-barrel (6Lo Leggio L. Kalogiannis S. Bhat M.K. Pickersgill R.W. Proteins. 1999; 36: 295-306Crossref PubMed Scopus (78) Google Scholar, 7Harris G.W. Jenkins J.A. Connerton I. Cummings N. Lo Leggio L. Scott M. Hazlewood G.P. Laurie J.I. Gilbert H.J. Pickersgill R.W. Structure. 1994; 2: 1107-1116Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), whereas family 11 enzymes are β-jelly roll proteins (8Sidhu G. Withers S.G. Nguyen N.T. McIntosh L.P. Ziser L. Brayer G.D. Biochemistry. 1999; 38: 5346-5354Crossref PubMed Scopus (173) Google Scholar). Consistent with their "endo" mode of action, the substrate binding cleft of xylanases extends along the length of the proteins and can accommodate from four to seven xylose residues (9Biely P. Kratky Z. Vrsanska M. Eur. J. Biochem. 1981; 119: 559-564Crossref PubMed Scopus (100) Google Scholar, 10Charnock S.J. Spurway T.D. Xie H. Beylot M.H. Virden R. Warren R.A. Hazlewood G.P. Gilbert H.J. J. Biol. Chem. 1998; 273: 32187-32199Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Each region that accommodates xylose moieties are known as subsites, which are given a negative or positive number dependent on whether they bind the glycone or aglycone region of the substrate, respectively, with glycosidic bond cleavage occurring between the -1 and +1 subsites (11Davies G.J. Wilson K.S. Henrissat B. Biochem. J. 1997; 321: 557-559Crossref PubMed Scopus (852) Google Scholar). The structures of xylanases in complex with oligosaccharides and inhibitors have revealed detailed information on the interactions of these enzymes with their substrates in the proximal, -2 and -1 glycone subsites (12White A. Tull D. Johns K.W.S.G. Rose D.R. Nat. Struct. Biol. 1996; 3: 149-154Crossref PubMed Scopus (189) Google Scholar, 13Notenboom V. Williams S.J. Hoos R. Withers S.G. Rose D.R. Nat. Struct. Biol. 1998; 5: 812-818Crossref PubMed Scopus (113) Google Scholar). The data show that at the -1 subsite the O-3 of xylose makes a hydrogen bond with a highly conserved lysine and histidine, whereas O-2 interacts with an asparagine and a glutamate (which acts as the catalytic nucleophile) that are invariant in GH10 xylanases. At the -2 subsite the xylose interacts with residues that are also highly conserved in family 10 xylanases with the O-2 of the sugar hydrogen bonding to a glutamate and tryptophan, the O-3 to an asparagine, and the endocyclic oxygen to a lysine. There is a paucity of information, however, on the mechanism by which the aglycone region of the substrate binding cleft of these enzymes interacts with the xylan backbone, although the three-dimensional structure of Cellvibrio japonicus xylanase 10A (CjXyn10A) in complex with xylopentaose bound to subsites -1 to +4 has been described (7Harris G.W. Jenkins J.A. Connerton I. Cummings N. Lo Leggio L. Scott M. Hazlewood G.P. Laurie J.I. Gilbert H.J. Pickersgill R.W. Structure. 1994; 2: 1107-1116Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 15Leggio L.L. Jenkins J. Harris G.W. Pickersgill R.W. Proteins. 2000; 41: 362-373Crossref PubMed Scopus (34) Google Scholar). These studies showed that a highly conserved aromatic residue stacks against the xylose at the +1 subsite, and although hydrophobic stacking interactions at the +3 and +4 subsites were the primary mechanism of protein-substrate recognition, these amino acids are not invariant in GH10 glycoside hydrolases, suggesting that xylan binding in the distal aglycone region of this enzyme family is variable. One of the fundamental differences between the two major families of xylanases is that the GH11 enzymes hydrolyze unsubstituted regions of xylan, whereas the corresponding family 10 glycoside hydrolases are able to attack decorated forms of the polysaccharide (16Biely P. Vrsanska M. Tenkanen M. Kluepfel D. J. Biotechnol. 1997; 57: 151-166Crossref PubMed Scopus (496) Google Scholar). The mechanism by which the side chains of decorated xylans are accommodated in the active site of GH10 xylanases is largely unknown. Schmidt et al. (17Schmidt A. Gubitz G.M. Kratky C. Biochemistry. 1999; 38: 2403-2412Crossref PubMed Scopus (65) Google Scholar) solved the structure of a non-natural decorated xylooligosaccharide comprising 1,2-(4-deoxy-β-l-threo-hex-4-enopyranosyluronic acid)-β-1,4-d-xylotriose. Although the backbone xylose residues were evident at subsites -1 to -3, the side chain was not observed, presumably because it was highly disordered. To probe the structural basis for the capacity of GH10 xylanases to hydrolyze decorated substrates, we have analyzed the biochemical properties of a family 10 enzyme from Cellvibrio mixtus (CmXyn10B) and determined its crystal structure in complex with unsubstituted and decorated substrates at resolutions from 1.7 to 1.4 Å. CmXyn10B comprises an N-terminal signal peptide and a 360-residue GH10 catalytic module; the enzyme does not contain noncatalytic accessory modules typical of plant cell wall-degrading enzymes (18Fontes C.M. Gilbert H.J. Hazlewood G.P. Clarke J.H. Prates J.A. McKie V.A. Nagy T. Fernandes T.H. Ferreira L.M. Microbiology. 2000; 146: 1959-1967Crossref PubMed Scopus (58) Google Scholar) and is shown to be the most active GH10 xylanase described to date. Its capacity to bind decorated substrates is conferred partly by the exposure of O-2 and O-3 groups at selected glycone and aglycone subsites and partly through productive interactions with the 4-O-MeGlcA side chains. The data provided here and in the accompanying paper by Fujimoto et al. (19Fujimoto Z. Kaneko S. Kuno A. Kobayashi H. Kusakabe I. Mizuno H. J. Biol. Chem. 2004; 279: 9606-9614Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) focus on a GH10 xylanase that contains an N-terminal lectin domain (19Fujimoto Z. Kaneko S. Kuno A. Kobayashi H. Kusakabe I. Mizuno H. J. Biol. Chem. 2004; 279: 9606-9614Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), reveal complementarity in the binding of decorated substrates between the glycone and aglycone regions of the active site, and demonstrate how these binding modes mediate synergy with the xylan accessory enzyme, α-glucuronidase. Bacterial Strains, Culture Conditions, and Plasmids—The Escherichia coli strain TUNER:pLysS (Novagen) was used in this study. The bacterium was cultured in Luria broth (LB) at 37 °C with aeration unless otherwise stated. The plasmid pCF1 is a recombinant of pET21b, which encodes the mature form of CmXyn10B corresponding to residues 11-379. The xylanase gene xyn10B (GenBank™ accession number AF049493) was inserted into the pET vector at the NdeI and XhoI restriction sites such that the translation stop codon was removed and the gene was in-frame with the His6 tag encoding sequence supplied by the vector (18Fontes C.M. Gilbert H.J. Hazlewood G.P. Clarke J.H. Prates J.A. McKie V.A. Nagy T. Fernandes T.H. Ferreira L.M. Microbiology. 2000; 146: 1959-1967Crossref PubMed Scopus (58) Google Scholar). Thus recombinant CmXyn10B contains a C-terminal His6 tag. Generation of CmXyn10B Mutants—Derivatives of CmXyn10B were generated using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions, using pCF1 as template DNA and the appropriate primers. The complete sequences of the DNA encoding the xylanase mutants were determined using an ABI 377 DNA Sequenator employing T7 forward and reverse primers and the custom sequencing primers 5′-AACGGCGCGCGATGTACAAGTCG-3′ and 5′-CAGCTGGTGAAAGAAGTAGCGC-3′ to confirm that only the desired mutations had been introduced. During the course of this study errors in the original manual sequencing of xyn10B, which resulted in 12 amino acid changes in the encoded enzyme, were detected and a corrected version of this gene has now been submitted to GenBank™. Expression and Purification of CmXyn10B—E. coli strain TUNER:pLysS harboring pCF1 was cultured in LB supplemented with 50 μg ml-1 ampicillin (1000 ml in 2-liter conical baffle flasks) at 37 °C and 180 rpm to mid-exponential phase (A600 nm 0.6). The culture was cooled and incubated at 30 °C before expression of CmXyn10B was induced by the addition of isopropyl β-thiogalactopyranoside to a final concentration of 0.2 mm. The culture was incubated for a further 5 h. The cells were then harvested by centrifugation at 4500 × g for 10 min at 4 °C and resuspended in 1/40th volume 20 mm Tris/HCl buffer, pH 8.0, containing 300 mm NaCl. Cells were lysed by sonication and centrifuged (25,000 × g) for 15 min at 4 °C to produce cell-free extract. CmXyn10B was purified from cell-free extract by immobilized metal affinity chromatography as described previously (20Xie H. Bolam D.N. Nagy T. Szabo L. Cooper A. Simpson P.J. Lakey J.H. Williamson M.P. Gilbert H.J. Biochemistry. 2001; 40: 5700-5707Crossref PubMed Scopus (57) Google Scholar) using Talon™ resin (Clontech, Palo Alto, CA). The protein eluted from the matrix was dialyzed against 3 × 500 volumes of 10 mm Tris/HCl buffer, pH 8.0 (Buffer A) and was then applied to a 3-ml DEAE Tris-Acryl column (15 × 50 mm). CmXyn10B was eluted with a linear 0-500 mm NaCl gradient in Buffer A. The recovered protein was concentrated to ∼1 ml using a Vivaspin 10-kDa molecular mass cut-off centrifugal concentrator (VIVASCIENCE, Hannover, Germany) and then applied to a High Load™ Superdex™ 75 column (16 × 600 mm; Amersham Biosciences) and eluted in Buffer A containing 150 mm NaCl. The columns were run at 1 ml min-1 using the Bio-Rad Biologic system. The purity of the protein was evaluated by SDS-PAGE. Protein concentration was determined from the calculated molar extinction coefficient of the enzyme at 280 nm (67,850 m-1 cm-1). Preparation of Oligosaccharides—Unsubstituted xylooligosaccharides were purchased from Megazyme International (Bray, County Wicklow, Ireland) and arabinoxylotriose (AX3) in which arabinofuranose is α-1,3-linked to the internal xylose moiety in xylotriose was a kind gift from Satoshi Kaneko. Aldotetraouronic acid (MX3) in which 4-O-MeGlcA acid is α-1,2-linked to the nonreducing sugar of xylotriose was prepared as follows. Glucuronoxylan (1 g; Megazyme Int.) was digested to completion with 1 mg CmXyn10B in a 5-ml reaction in 50 mm sodium phosphate buffer, pH 7.0, at 37 °C for 18 h. MX3 was purified by Dowex X-100 (Sigma) chromatography in which the bound decorated oligosaccharide was eluted with a 0-500 mm sodium chloride gradient in 50 mm sodium phosphate buffer, pH 7.0. The MX3 was then further purified by size exclusion chromatography using a Bio-gel P2 column (2.5 × 100 cm) in which the oligosaccharide was eluted in distilled water at a flow rate of 0.2 ml min-1. The identity of the product as MX3 was verified by digestion with the α-glucuronidase GlcA67A from C. japonicus (21Nagy T. Emami K. Fontes C.M. Ferreira L.M. Humphry D.R. Gilbert H.J. J. Bacteriol. 2002; 184: 4925-4929Crossref PubMed Scopus (43) Google Scholar) followed by Dionex-HPLC analysis, which revealed xylotriose and 4-O-MeGlcA as the reaction products. Arabinoxylobiose (AX2), in which the arabinofuranose is α-1,3-linked to the nonreducing xylose unit in xylobiose, was prepared as follows. Wheat arabinoxylan (1 g; Megazyme Int.) was digested to completion with 1 mg of CmXyn10B in a 5-ml reaction in 50 mm sodium phosphate buffer, pH 7.0, at 37 °C for 18 h. AX2 was purified by size exclusion chromatography as described above. The identity of the product as AX2 was verified by digestion with the arabinofuranosidase Abf51A from C. japonicus (22Beylot M.H. McKie V.A. Voragen A.G. Doeswijk-Voragen C.H. Gilbert H.J. Biochem. J. 2001; 358: 607-614Crossref PubMed Scopus (59) Google Scholar) and Dionex-HPLC analysis, which revealed xylobiose and arabinose as the reaction products. Enzyme Assays—The activity of the GH10 xylanases against aryl β-glycosides was determined as described previously (23Charnock S.J. Lakey J.H. Virden R. Hughes N. Sinnott M.L. Hazle- wood G.P. Pickersgill R. Gilbert H.J. J. Biol. Chem. 1997; 272: 2942-2951Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Xylanase activity was performed essentially as described by Charnock et al. (23Charnock S.J. Lakey J.H. Virden R. Hughes N. Sinnott M.L. Hazle- wood G.P. Pickersgill R. Gilbert H.J. J. Biol. Chem. 1997; 272: 2942-2951Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) except that the release of reducing sugar was determined using the Somogyi-Nelson reagent (24Somogyi M. J. Biol. Chem. 1952; 195: 19-23Abstract Full Text PDF PubMed Google Scholar). Each assay was performed in triplicate. To evaluate the activity of the two xylanases against xylooligosaccharides, progress curves were carried out as described previously, and the data were used to determine kcat/Km following the equation of Matsui et al. (25Matsui I. Ishikawa K. Matsui E. Miyairi S. Fukui S. Honda K. J. Biochem. (Tokyo). 1991; 109: 566-569Crossref PubMed Scopus (74) Google Scholar). The bond cleavage frequency and kcat/Km data obtained from these experiments were used to calculate the ΔG of xylose binding at each of the subsites following the method of Suganuma et al. (26Suganuma T. Matsuno R. Ohnishi M. Hiromi K. J. Biochem. (Tokyo). 1978; 84: 293-316Crossref PubMed Scopus (136) Google Scholar). Crystallization and Data Collection—Pure proteins, as judged by SDS-PAGE, were washed into water by repeated dilution (40 volumes of water) and concentrated in a Vivaspin 10-kDa concentrator. The E262S nucleophile mutant of CmXyn10B was screened by the hanging drop vapor diffusion method using the PEG/Ion and Crystal screens (Hampton Research, Aliso Viejo, CA). Crystallization conditions were optimized independently for each xylooligosaccharide complex. Crystals grew in ∼2-4 days at 18 °C. Drops contained 1 or 2 μl of protein (25 mg ml-1), 0.5 or 1 μl of xylooligosaccharide (10 mm), and 1 μl of mother liquor. Crystallization conditions for each xylooligosaccharide complex were as follows: xylopentaose, 0.20 m MgCl2, 0.1 m Tris, pH 8.0, and 30% PEG 4000; AX2, 0.35 m MgCl2, 0.1 m Tris, pH 6.5, 30% PEG 4000, and 5% PEG 400; AX3, 0.20 m MgCl2, 0.1 m Tris, pH 8.5, 30% PEG 4000, and 5% PEG 400; MX3, 0.10 m MgCl2, 0.1 m Tris, pH 6.5, 30% PEG 4000, and 5% isopropanol. Crystals in the form of plates were harvested in rayon fiber loops and cryoprotected in 15% 2-methyl-2,4-pentanediol, 85% mother liquor prior to flash freezing in liquid N2. All data were collected from single crystals at 120 K, over an oscillation range of 135° with a Δϕ of 0.5°. Data for CmXyn10B E262S in complex with xylopentaose were collected in the home laboratory using a MarResearch image plate detector on a Rigaku rotating anode RU-200 x-ray generator with a Cu target operating at 50 kV and 100 mA and focusing x-ray optics (Osmics). Data for CmXyn10B E262S in complex with AX3 were collected at the Daresbury Synchrotron Radiation Source (SRS) on beamline PX-14.2. Data for CmXyn10B E262S in complex with AX2 and MX3 were collected on the European Synchrotron Radiation Facility (ESRF) beamline ID-29. SRS and ESRF data were collected using ADSC Quantum-4 and Quantum-210 charge-coupled device detectors, respectively. Structure Solution and Refinement—All data were processed and scaled with the HKL suite (27Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar). All other computing used the CCP4 suite (28Collaborative Computational Project Number 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar) unless otherwise stated. All crystals belonged to space group P212121 with approximate unit cell dimensions a = 47 Å, b = 68 Å, and c = 105 Å. The structure of CmXyn10B E262S in complex with xylopentaose was solved by molecular replacement with the program AMoRe and with data in the resolution range 20-3.0 Å and an outer radius of Patterson integration of 25 Å. The protein atoms from the catalytic core domain of the C. japonicus Xyn10A (protein data bank 1clx) was used as the search model. AMoRe generated one solution corresponding to one molecule in the asymmetric unit. This gives a VM of 2.0 A3 D-1 and a solvent content of 36.6%. Prior to refinement and model building, 5% of the observations were set aside for cross-validation analysis (29Brünger A.T. Nature. 1992; 355: 472-475Crossref PubMed Scopus (3872) Google Scholar) and were used to monitor various refinement strategies such as the weighting of geometrical and temperature factor restraints as well as the insertion of solvent water during maximum likelihood refinement. REFMAC and ARP/wARP (CCP4 suite) were used to build the correct sequence into electron density automatically. Manual corrections of the model using the X-FIT routines of the program QUANTA (Accelrys) were interspersed with cycles of maximum likelihood refinement using REFMAC. Solvent molecules were added automatically using ARP/wARP and inspected manually prior to deposition. The refined structure of the CmXyn10B E262S complexed with xylopentaose was used as the starting model for the refinement of the decorated oligosaccharide complexes. The same 5% of observations was maintained as the Rfree set in each case. Ideal coordinates for stereochemical dictionaries for the substituted xylooligosaccharides were generated using the energy minimization CHARMm function in QUANTA. Structures were validated using PROCHECK (30Laskowski R.A. McArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 282-291Crossref Google Scholar). Coordinates have been deposited with the Macromolecular Structures Database (Table I).Table IRefinement and structure quality statistics for the Cellvibrio mixtus Xyn10B mutant (E262S) complex structuresXylopentaoseAX2AX3MX3Resolution of data (outer shell), Å20−1.72 (1.78−1.72)35−1.43 (1.48−1.43)30−1.6 (1.66−1.6)35−1.55 (1.61−1.55)R-mergeaR-merge=∑hklΣi/Ihkli-〈Ihkl〉/∑hklΣi/Ihkli. (outer shell)0.038 (0.174)0.055 (0.334)0.076 (0.432)0.057 (0.358)Mean I/σI (outer shell)46.0 (10.35)26.02 (1.94)14.6 (1.93)30.22 (3.39)Completeness (outer shell), %99.6 (98.7)83.3 (20.5)98.3 (90.3)95.4 (71.0)Multiplicity (outer shell)6.02 (5.49)5.62 (1.38)3.25 (2.23)5.53 (2.25)No. unique reflections34539494174205045059No. rejections21320603133333No. protein atoms2819285828692841No. ligand atoms67275669No. solvent waters561569591583Rcryst0.1400.1510.1520.158Rfree0.1860.1860.1890.199r.m.s. deviation 1-2 bond (Å)0.0140.0140.0100.011r.m.s. deviation 1-3 angle (°)1.5391.6091.3191.333r.m.s. deviation chiral volume (Å3)0.1020.0980.0830.097Avg. main chain B (Å2)12.9710.6812.299.27Avg. side chain B (Å2)15.3713.2213.9211.31Avg. substrate B (Å2)20.9616.6518.43717.49Avg. solvent B (Å2)28.3925.0529.9124.03PDB code1uqy1ur11ur21uqza R-merge=∑hklΣi/Ihkli-〈Ihkl〉/∑hklΣi/Ihkli. Open table in a new tab Biochemical Properties of CmXyn10B—CmXyn10B was purified to electrophoretic homogeneity, and its biochemical properties were determined (Tables II and III). The enzyme displays catalytic properties typical of a GH10 xylanase. The activity of CmXyn10B against xylooligosaccharides increases with the degree of polymerization (d.p.) of the substrate, although the difference in the hydrolysis rate between xylohexaose and xylopentaose is modest, implying that the sixth binding site (+4 subsite) interacts only weakly with the oligosaccharide substrate. The hydrolysis of xylotetraose exclusively into xylobiose demonstrates that the enzyme contains two subsites on either side of the point of cleavage, but as no xylotriose was generated, the xylanase does not contain a significant -3 subsite (Fig. 1). The catalytic efficiency of CmXyn10B and its mode of action against different xylooligosaccharides and aryl-β-xylosides (Table IV) shows that the -2 and +2 subsites have binding energies of 9.0 and 3.7 kcal mol-1, respectively. The maximum binding energy at the +3 is 1.1 kcal mol-1, assuming that the increased activity of CmXyn10B against xylopentaose, compared with xylotetraose, is due entirely to binding at this location.Table IIBiochemical properties of CjXyn10A and CjXyn10CEnzymekcat/Kmakcat/Km was determined by measuring progress curves at a substrate concentration below Km.Specific activitybSpecific activity is expressed as mol of product produced/mol of enzyme/min.X3X4X5X6PNPX2PNPXPNPG2OXRAXWAXGXmin−1 M−1min−1CmXyn10B6.0 × 1043.2 × 1072.0 × 1082.7 × 1088.2 × 1072.1 × 1022.4 × 104114205882799617782F340A1.3 × 1037.1 × 105—6.2 × 1061.5 × 107—1.7 × 104————Y200F—2.8 × 107—2.3 × 1082.7 × 1061.0 × 1021.3 × 1048823262736526347E262S———NDND—NDNDNDNDNDCjXyn10A9.2 × 1037.3 × 1051.3 × 1071.2 × 1085.1 × 1062.9 × 1013.2 × 1039887948331226CjXyn10CcThe biochemical properties of CjXyn10C and CfXyn10A have been reported elsewhere by Pell et al.3 and in Ref. 10, respectively.7.3 × 1028.5 × 1042.9 × 1061.0 × 1072.3 × 1063.4 × 1011.2 × 1011159122314971359CfXyn10AcThe biochemical properties of CjXyn10C and CfXyn10A have been reported elsewhere by Pell et al.3 and in Ref. 10, respectively.2.5 × 1041.8 × 1071.1 × 1081.9 × 1081.5 × 1085.2 × 1026.2 × 105615———a kcat/Km was determined by measuring progress curves at a substrate concentration below Km.b Specific activity is expressed as mol of product produced/mol of enzyme/min.c The biochemical properties of CjXyn10C and CfXyn10A have been reported elsewhere by Pell et al.3 and in Ref. 10Charnock S.J. Spurway T.D. Xie H. Beylot M.H. Virden R. Warren R.A. Hazlewood G.P. Gilbert H.J. J. Biol. Chem. 1998; 273: 32187-32199Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, respectively. Open table in a new tab Table IIIKinetic constants of CmXyn10B and the mutant F340A against xylansCmXyn10BSubstrateaGX, glucuronoxylan; OX, oat spelt xylan.KmkcatKm/kcatmg/ml−1min−1min−1 mg−1 mlWild typeGX3.7912,3643,262Wild typeOX6.1719,9003,225F340AGX13.44,235316a GX, glucuronoxylan; OX, oat spelt xylan. Open table in a new tab Table IVBinding energies at the subsites of CmXyn10BΔG − 2 subsiteΔG + 2 subsiteΔG + 3 subsitekcal mol−19.0 (PNPX2 and PNPX)3.7 (X42 and X31)1.1 (X42 and X53) Open table in a new tab Against both poorly and highly decorated xylans, CmXyn10B initially generates a range of oligosaccharides that progressively become smaller in size as the enzyme reactions progress (data not shown). Against both wheat arabinoxylan and glucuronoxylan, the unsubstituted end products of the reactions comprise xylose and xylobiose. An oligosaccharide that did not co-migrate with linear xylooligosaccharides was also
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