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• W2065905475 abstract "Reducing end xylose-releasing exo-oligoxylanase from Bacillus halodurans C-125 (Rex) hydrolyzes xylooligosaccharides whose degree of polymerization is greater than or equal to 3, releasing the xylose unit at the reducing end. It is a unique exo-type glycoside hydrolase that recognizes the xylose unit at the reducing end in a very strict manner, even discriminating the β-anomeric hydroxyl configuration from the α-anomer or 1-deoxyxylose. We have determined the crystal structures of Rex in unliganded and complex forms at 1.35–2.20-Å resolution and revealed the structural aspects of its three subsites ranging from –2 to +1. The structure of Rex was compared with those of endo-type enzymes in glycoside hydrolase subfamily 8a (GH-8a). The catalytic machinery of Rex is basically conserved with other GH-8a enzymes. However, subsite +2 is blocked by a barrier formed by a kink in the loop before helix α10. His-319 in this loop forms a direct hydrogen bond with the β-hydroxyl of xylose at subsite +1, contributing to the specific recognition of anomers at the reducing end. Reducing end xylose-releasing exo-oligoxylanase from Bacillus halodurans C-125 (Rex) hydrolyzes xylooligosaccharides whose degree of polymerization is greater than or equal to 3, releasing the xylose unit at the reducing end. It is a unique exo-type glycoside hydrolase that recognizes the xylose unit at the reducing end in a very strict manner, even discriminating the β-anomeric hydroxyl configuration from the α-anomer or 1-deoxyxylose. We have determined the crystal structures of Rex in unliganded and complex forms at 1.35–2.20-Å resolution and revealed the structural aspects of its three subsites ranging from –2 to +1. The structure of Rex was compared with those of endo-type enzymes in glycoside hydrolase subfamily 8a (GH-8a). The catalytic machinery of Rex is basically conserved with other GH-8a enzymes. However, subsite +2 is blocked by a barrier formed by a kink in the loop before helix α10. His-319 in this loop forms a direct hydrogen bond with the β-hydroxyl of xylose at subsite +1, contributing to the specific recognition of anomers at the reducing end. Glycoside hydrolases (GHs) 1The abbreviations used are: GH, glycoside hydrolase; CBH, cellobiohydrolase; Xn, xylooligosaccharide whose degree of polymerization is n; X3-de, deoxy-X3; HPLC, high pressure liquid chromatography; WT, wild type. 1The abbreviations used are: GH, glycoside hydrolase; CBH, cellobiohydrolase; Xn, xylooligosaccharide whose degree of polymerization is n; X3-de, deoxy-X3; HPLC, high pressure liquid chromatography; WT, wild type. can be grouped into two broad classes, exo- and endo-enzymes, according to how they degrade polysaccharides. Endo-GHs cleave glycosidic bonds randomly in the interior of the polysaccharide chain, whereas exo-GHs cleave mono-, di-, or oligosaccharide units from one end of the chain. Branched polysaccharides have more nonreducing ends than reducing ends, and most currently identified exo-GHs are ones acting on the nonreducing ends of polysaccharides.Rex (reducing end xylose-releasing exo-oligoxylanase) from Bacillus halodurans C-125 (gene product of BH2105) is one of the few exo-type enzymes that act on the reducing end (1Honda Y. Kitaoka M. J. Biol. Chem. 2004; 279: 55097-55103Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Unlike other so-called “reducing end-specific” exo-GHs, such as MalZ (maltodextrin glucosidase) from Escherichia coli K12 (2Tapio S. Yeh F. Shuman H.A. Boos W. J. Biol. Chem. 1991; 266: 19450-19458Abstract Full Text PDF PubMed Google Scholar), an amylolytic enzyme from Thermotoga maritima (TM1835) (3Lee M.H. Kim Y.W. Kim T.J. Park C.S. Kim J.W. Moon T.W. Park K.H. Biochem. Biophys. Res. Commun. 2002; 295: 818-825Crossref PubMed Scopus (32) Google Scholar), oligoxyloglucan reducing end-specific cellobiohydrolase (4Yaoi K. Mitsuishi Y. J. Biol. Chem. 2002; 277: 48276-48281Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), and reducing end-specific processive cellobiohydrolases (CBHs) (5Barr B.K. Hsieh Y.L. Ganem B. Wilson D.B. Biochemistry. 1996; 35: 586-592Crossref PubMed Scopus (232) Google Scholar), Rex recognizes the xylose unit of the reducing end in a highly strict manner, even discriminating the β-anomeric hydroxyl configuration from the α-anomer or 1-deoxyxylose. To our knowledge, therefore, Rex is the only complete exo-GH that releases a monosaccharide from the reducing end. The enzyme is thought to play a key role in the intracellular xylan metabolism of B. halodurans by cleaving xylooligosaccharides. Considering the substrate specificity for xylooligosaccharides of various lengths, the enzyme is presumed to have three subsites (–2, –1, and +1). Because Rex is an inverting enzyme that produces the α-anomer at the reducing end, spontaneous mutarotation of the substrate is required for subsequent processing in vitro. It remains unclear whether there are any factors supporting the mutarotation of xylooligosaccharides in vivo or if there are any biological reasons for such an inefficient processing mechanism.Rex belongs to the GH-8 family according to the CAZy classification (available on the World Wide Web at afmb.cnrs-mrs.fr/CAZY/) (6Henrissat B. Biochem. J. 1991; 280: 309-316Crossref PubMed Scopus (2589) Google Scholar). GH-8 and GH-48 are grouped to form clan GH-M, sharing similar (α/α)6 barrel fold (7Guimarães B.G. Souchon H. Lytle B.L. David Wu J.H. Alzari P.M. J. Mol. Biol. 2002; 320: 587-596Crossref PubMed Scopus (80) Google Scholar). Within the GH-8 family, the crystal structures of three extracellular endo-GHs, CelA (endoglucanase from Clostridium thermocellum) (8Alzari P.M. Souchon H. Dominguez R. Structure. 1996; 4: 265-275Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), pXyl (a cold-adapted xylanase from the Antarctic bacterium Pseudoalteromonas haloplanktis) (9Van Petegem F. Collins T. Meuwis M.A. Gerday C. Feller G. Van Beeumen J. J. Biol. Chem. 2003; 278: 7531-7539Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), and ChoK (chitosanase from Bacillus sp. K17) (10Adachi W. Sakihama Y. Shimizu S. Sunami T. Fukazawa T. Suzuki M. Yatsunami R. Nakamura S. Takénaka A. J. Mol. Biol. 2004; 343: 785-795Crossref PubMed Scopus (99) Google Scholar), have been determined. In particular, the subsites of CelA (–3 to +3) have definitely been determined with the complex structure with cellopentaose at atomic resolution (11Guérin D.M. Lascombe M.B. Costabel M. Souchon H. Lamzin V. Béguin P. Alzari P.M. J. Mol. Biol. 2002; 316: 1061-1069Crossref PubMed Scopus (121) Google Scholar). Adachi et al. (10Adachi W. Sakihama Y. Shimizu S. Sunami T. Fukazawa T. Suzuki M. Yatsunami R. Nakamura S. Takénaka A. J. Mol. Biol. 2004; 343: 785-795Crossref PubMed Scopus (99) Google Scholar) proposed dividing the GH-8 family into three subfamilies (GH-8a, -8b, and -8c), depending on the position of the catalytic base residue. CelA, pXyl, and Rex are grouped into the GH-8a subfamily, and ChoK is grouped into GH-8b. GH-8a enzymes have proton donor and catalytic base residues at the N termini of the α4 and α8 helices within the (α/α)6 barrel, respectively. The two catalytic residues of Rex (Glu70 as a proton donor and Asp263 as a catalytic base) have already been confirmed by site-directed mutagenesis (1Honda Y. Kitaoka M. J. Biol. Chem. 2004; 279: 55097-55103Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Interestingly, the catalytic base of GH-8a enzymes (Asp) is replaced by Asn (or Ser) in the GH-8b and -8c enzymes. The crystal structure of ChoK revealed that the catalytic base of GH-8b is located in the long loop inserted between α7 and α8 (10Adachi W. Sakihama Y. Shimizu S. Sunami T. Fukazawa T. Suzuki M. Yatsunami R. Nakamura S. Takénaka A. J. Mol. Biol. 2004; 343: 785-795Crossref PubMed Scopus (99) Google Scholar). We describe here crystal structures of Rex and show the structural basis for its strict substrate specificity, especially for a xylose unit at the reducing end.EXPERIMENTAL PROCEDURESCrystallography—Expression and purification of the wild-type and E70A mutant enzymes were previously reported (1Honda Y. Kitaoka M. J. Biol. Chem. 2004; 279: 55097-55103Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Both types of enzyme were crystallized under the conditions described elsewhere (12Honda Y. Fushinobu S. Hidaka M. Wakagi T. Shoun H. Kitaoka M. Acta Crystallogr. Sect. F. 2005; 61: 291-292Crossref PubMed Scopus (7) Google Scholar). The xylose complex (WT-xylose) was prepared by co-crystallization using a reservoir solution containing 10 mm xylose. The xylobiose complex of the E70A mutant enzyme (E70A-xylobiose) was prepared by co-crystallization using a reservoir solution containing 10 mm xylotriose, and the crystals that grew in 6 days were used for data collection. Diffraction data were collected using a charge-coupled device camera on the BL-5A station at the Photon Factory and the NW-12 station at the Photon Factory AR, High Energy Accelerator Research Organization (KEK), Tsukuba, Japan. The crystals were flash-cooled in a stream of liquid nitrogen at 100 K. Diffraction images were indexed, integrated, and scaled using the HKL2000 program suite (13Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38355) Google Scholar). Initial phases of the native structure were obtained by the molecular replacement method, using the structure of psychrophilic endo-β-1,4-xylanase from P. haloplanktis (Protein Data Bank code 1H14) as a search model. Molecular replacement was performed with MOLREP (14Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4120) Google Scholar) in the CCP4 program suite (15Project Collaborative Computing Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19703) Google Scholar). The program ARP/wARP (16Morris R.J. Perrakis A. Lamzin V.S. Acta Crystallogr. Sect. D. 2002; 58: 968-975Crossref PubMed Scopus (221) Google Scholar) was used for automatic model building. Visual inspection of the models was performed using XtalView (17McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2019) Google Scholar). Crystallographic refinement was carried out using CNS1.1 (18Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J-S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D. 1998; 54: 905-921Crossref PubMed Scopus (16929) Google Scholar). The data collection and refinement statistics are summarized in Table I. The complex structures were solved by starting from the refined native structure. The figures were prepared using SPOCK (19Christopher J.A. Baldwin T.O. J. Mol. Graphics Model. 1998; 16: 285Google Scholar), Raster3D (20Merritt E.A. Bacon D.J. Methods Enzymol. 1997; 277: 505-524Crossref PubMed Scopus (3869) Google Scholar), MOLSCRIPT (21Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar), and XtalView.Table ICrystallographic data collection and refinement statisticsWT-nativeWT-xyloseE70A-xylobioseData collection statisticsX-ray sourcePF BL-5APF BL-5APF-AR NW-12Wavelength (Å)1.0001.0001.000Space groupP212121P212121P212121Unit-cell parametersa (Å)52.6952.3053.30b (Å)86.0285.4286.61c (Å)87.9287.4487.71Resolution (Å)62.02 to 1.35 (1.40 to 1.35)62.86 to 2.20 (2.28 to 2.20)62.02 to 1.45 (1.50 to 1.45)Measured reflections1,797,205687,303891,274Unique reflections87,06920,27272,191Completeness (%)98.4 (96.4)99.8 (99.3)99.2 (97.9)Mean I/σ (I)36.0 (4.7)24.1 (6.4)26.5 (3.9)RmergeaRmerge = ΣhΣiI(h,i) — 〈I(h)〈|/ΣhΣiI(h,i), where I(h,i) is the intensity of the ith measurement of reflection h and 〈I(h)〉 is the average value over multiple measurements. (%)5.3 (28.5)8.4 (24.4)5.3 (29.5)Refinement statisticsResolution (Å)25.19-1.3544.88-2.2031.39-1.45R/RfreebCalculated using a test data set; 5% of total data randomly selected from the observed reflections. (%)18.0/19.917.1/21.117.1/18.0No. of waters583302495Average B-factors (Å2)Protein13.8421.4712.44Waters28.8431.0630.38Ni2+10.2017.548.99Glycerol (packing site)16.7923.3713.02Ligand 130.62 (xylose)10.72 (xylobiose)Ligand 221.27 (glycerol)Root mean square deviation from ideal valuesBond lengths (Å)0.0050.0060.005Bond angles (degrees)1.21.21.3a Rmerge = ΣhΣiI(h,i) — 〈I(h)〈|/ΣhΣiI(h,i), where I(h,i) is the intensity of the ith measurement of reflection h and 〈I(h)〉 is the average value over multiple measurements.b Calculated using a test data set; 5% of total data randomly selected from the observed reflections. Open table in a new tab Site-directed Mutagenesis and Enzyme Assay—Site-directed mutagenesis for H319A was performed using the PCR overlap extension method (22Higuchi R. Krummel B. Saiki R.K. Nucleic Acids Res. 1988; 16: 7351-7367Crossref PubMed Scopus (2087) Google Scholar). The following mutagenic oligonucleotide primer was used (the mismatched bases are underlined): 5′-GAG AAA TCA TTG GCC CCT GTC GGA CTG AT-3′. Preparation, purification, and activity measurement of the mutant enzyme were carried out as described previously (1Honda Y. Kitaoka M. J. Biol. Chem. 2004; 279: 55097-55103Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). As to the HPLC analysis of the anomeric form of the products, the concentrations of the substrate (X3) and the enzyme (H319A mutant) were 50 mm and 19.3 μm, respectively.RESULTSCrystallography—The unliganded crystal structure of the wild-type Rex (WT-native) was solved by means of molecular replacement, using the crystal structure of pXyl. The complex structure of the wild-type Rex with xylose (WT-xylose) was determined by a co-crystallization method. In the WT-xylose structure, the electron density of a xylose molecule was found at subsite +1, following the definition of the subsites of CelA (Fig. 1a). Although the density peaks were relatively low and the temperature factors were relatively high (Table I), the density was clear in shape, so we could confidently determine the orientation and conformation of the xylose molecule. Further attempts to obtain other types of complex structures (i.e. co-crystallization of the wild-type Rex in 10 mm xylobiose or a mixture of 5 mm xylose and 5 mm xylobiose) resulted in a similar density to that in the case of WT-xylose (a xylose molecule was bound at subsite +1; data not shown). When the wild-type Rex crystals were soaked in 10 mm xylotriose for a short time (10 s), density peaks for sugar units ranging from subsite –2 to +1 were observed. However, the density at subsite –1 was ambiguous and appeared to reflect a mixture of several conformations, probably involving a fraction of the cleaved substrate. The second type of complex structure was obtained by co-crystallizing the inactive E70A mutant with xylotriose (E70A-xylobiose). In the substrate binding cleft of E70A-xylobiose, the clear density of a xylobiose unit at subsites –2 and –1, as well as a glycerol at subsite +1, was observed (Fig. 1b). There are two possibilities that explain the discrepancy between the co-crystallized reagent (xylotriose) and the density observed for xylobiose: (a) xylotriose had been cleaved during the crystal formation (6 days), although the hydrolytic activity of the E70A mutant is 10–4 orders lower that of the wild-type; and (b) xylotriose is bound at subsites –3 to –1, but a xylose moiety at the nonreducing end could not be detected due to high mobility or disorder.All three structures contain a metal ion and a glycerol, both of which bind at a crystal packing interface far from the active site (Fig. 2b). The metal ion was assigned as a nickel ion (Ni2+) because a Ni2+-nitrilotriacetic acid-agarose column was used for purification, and no other candidate was included in the reservoir solution. Refined temperature factors of the nickel ion in the three structures were within 8.9–17.6 Å2 (Table I), and the refined Fo – Fc maps were almost flat (data not shown). The nickel ion was tetrahedrally coordinated by Gln27, Gln30, Asp253′, and His259′ (symmetry-related residues are indicated by primes). The glycerol molecule was held by the main-chain atoms of Gln192, Tyr247, and Asp248 and the side chain of Trp123′. Glycerol was absolutely required for crystallization (12Honda Y. Fushinobu S. Hidaka M. Wakagi T. Shoun H. Kitaoka M. Acta Crystallogr. Sect. F. 2005; 61: 291-292Crossref PubMed Scopus (7) Google Scholar). A polypeptide chain extending from Glu6 and Pro381 was modeled in WT-xylose and E70A-xylobiose, whereas Glu45 and Thr46 were not included in WT-native because of local disorder.Fig. 2Ribbon diagrams of the GH-8a enzymes. The catalytic residues, ligand molecules, and metal ions are shown as black sticks, a ball-and-stick model, and spheres, respectively. a, side view of the (α/α)6 barrel of Rex. b, top view of the barrel in a. The position of the α10 helix is indicated. c, top view of the barrel of the wild-type pXyl complexed with a xylose at subsite +4. The side chain of the catalytic proton donor (Glu78) is positioned differently from in the other two enzymes. d, top view of the barrel of CelA. A part of the cellopentaose molecule (subsites –3 to –1 out of –3 to +2) and the cellotriose molecule (subsites +1 to +3) are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Overall Structure—The structure of Rex comprises a disordered (α/α)6 barrel similar to that of pXyl, whereas CelA has a less disordered (α/α)6 barrel with a circular cross-section (Fig. 2). The root mean square deviations as to pXyl and CelA are 1.8 Å for 357 residues and 2.3 Å for 335 residues, respectively. There are four free cysteine residues located inside the molecule. This characteristic is usual for intracellular enzymes. On the other hand, two cysteine residues of pXyl, which is an extracellular enzyme, form a disulfide bond (9Van Petegem F. Collins T. Meuwis M.A. Gerday C. Feller G. Van Beeumen J. J. Biol. Chem. 2003; 278: 7531-7539Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). The molecular surfaces of the three GH-8a enzymes are shown in Fig. 3. The substrate-binding cleft of CelA is clearly larger than those of the other two enzymes. Interestingly, subsite +2 of Rex is blocked by a barrier at the upper side of the cleft (described below). In contrast, the other two endo-GHs have a long cleft spanning the molecule, which can accommodate a long polysaccharide chain.Fig. 3Molecular surfaces of Rex (a), pXyl (b), and CelA (c), showing the substrate binding cleft. Positive and negative potentials are shown in blue and red, respectively. Ligand oligosaccharides are shown as a ball-and-stick model. c, a part of the cellopentaose molecule (subsites –3 to –1 of –3 to +2) and the cellotriose molecule (subsites +1 to +3) are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Complex Structures—WT-native and the two complex structures (WT-xylose and E70A-xylobiose) of Rex were almost identical (root mean square deviation for Cα atoms <0.26 Å and for all atoms <0.44 Å between all pairs of the three Rex structures). There was no large conformational change on substrate binding like on other GH-8a enzymes. For example, substrate binding on CelA induces only small structural changes in the protein, mostly slight reorientations of aromatic and polar side chains in contact with the substrate (11Guérin D.M. Lascombe M.B. Costabel M. Souchon H. Lamzin V. Béguin P. Alzari P.M. J. Mol. Biol. 2002; 316: 1061-1069Crossref PubMed Scopus (121) Google Scholar). However, substrate binding on Rex induced slight movements in two regions (Thr62–Asn64 and Gly355–Arg357) around subsite +1 (Fig. 4a). The main chain moves toward the substrate in both of the complex structures. In the WT-xylose structure, the side chain of Arg357 swings to form a specific interaction with the xylose at subsite +1, accompanied by movement of the adjacent Asn356 residue. The side chain of Arg357 was ordered in WT-xylose, whereas it was disordered in the other two structures.Fig. 4The subsites of Rex and GH-8a enzymes.a, stereoview of superimposition of the three structures of Rex around the substrate-binding site. The WT-native, WT-xylose, and E70A-xylobiose structures are shown in yellow, gray, and green, respectively. b, stereoview of superimpositioning of Rex (gray), pXyl (green), and CelA (yellow). The structures were superimposed using the atoms of the two catalytic residues and the third catalytically important residue (Asp128/Asp144/Asp152 in Rex/pXyl/CelA). The xylobiose (–2 to –1) and xylose (+1) in Rex, the xylose in pXyl (+4), and the cellopentaose in CelA (subsites –3 to +2) are shown as a ball-and-stick model. The partially bound product molecule in CelA (cellotriose at subsites +1to +3) is shown as thin sticks. Residue names and numbers are labeled in the order of Rex/pXyl/CelA with each color.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The xylose molecule in WT-xylose takes on the 4C1 conformation, and all of the oxygen atoms (O-1 to O-5) form direct or water-mediated hydrogen bonds with protein atoms (Fig. 5a). His319 forms one of the two direct hydrogen bonds present between xylose and the Rex protein, which recognizes the β-hydroxyl group at the O-1 position. A stacking interaction of Tyr360 with the β-face of the xylose also occurs. This residue can sterically interfere binding of an α-anomer. When an α-glucose molecule was superimposed onto the β-xylose, the α-anomeric hydroxyl oxygen atom was located at distances of 2.7 and 2.5 Å from the Oβ and Oγ atoms of Tyr360, respectively (see Supplemental Material). Actually, electron density for only the β-anomer was observed, probably because of these interactions.Fig. 5Schematic drawing of the active sites in the WT-xylose (a) and E70A-xylobiose (b) structures.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In the E70A-xylobiose complex, the xylobiose molecule takes on the 4C1 conformation at both of the two sugar rings and the β-anomeric configuration at the reducing end. The glycerol molecule is located at almost the same position as subsite +1 and forms a water-mediated hydrogen bond with the xylobiose (Fig. 5b). Although the positions of the three hydroxyl oxygen atoms of the glycerol are slightly different from those of xylose at subsite +1, they interact with several residues at subsite +1 and cause movement of the main chain in the two regions described above. All of the oxygen atoms form direct or water-mediated hydrogen bonds with the protein, and the xylose ring at subsite –2 of the xylobiose is stacked with Trp112 at the β-face.Subsites—Fig. 4b shows composite superimpositioning of the subsites of the three GH-8a enzymes: the WT-xylose structure of Rex combined with xylobiose in the E70A-xylobiose structure (gray), the pXyl D144N mutant structure (Protein Data Bank code 1H14) combined with a xylose molecule at subsite +4 in the complex structure (Protein Data Bank code 1H12) (green), and the CelA E95Q mutant structure complexed with cellopentaose (Protein Data Bank code 1KWF) (yellow). Both the cellopentaose molecule (subsites –3 to +2; ball-and-stick model) and the partially bound product molecule (cellotriose at subsites +1to +3) are shown. Xylose and xylobiose in Rex approximately overlap with the glucose units at subsites –2 to +1 in CelA.At subsite –3 of CelA, Trp205 forms a stacking interaction, and the side chain of Arg204 forms a hydrogen bond with the O-3 atom. However, there is no subsite here in pXyl and Rex. In Rex, the Trp is substituted by Ile188, and there is no corresponding residue with the Arg, losing specific interactions to a sugar moiety at this site. Rex exhibits significantly higher Km for xylooligasaccharides of longer than xylotetraose (1Honda Y. Kitaoka M. J. Biol. Chem. 2004; 279: 55097-55103Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). There is, however, no steric hindrance at subsite –3, and the subsite is highly accessible to solvent. We could not find any convincing structural factor that can make the binding effect on this subsite negative. Perhaps the side chain of Glu190, which is solvent-exposed and somewhat disordered in the crystal structure, may interfere with the sugar binding in an extended conformation (Fig. 4b). At subsite –2, the bound xylose/glucose groups of Rex and CelA almost completely overlap. The stacking Trp residue (Trp112/Trp124/Trp132 in Rex, pXyl, and CelA) is conserved in GH-8a enzymes. The O-2, O-3, and O-5 atoms at subsite –2 form water-mediated hydrogen bonds with Tyr244 and Tyr198 (Fig. 5b).A significant kink is observed between sugar rings at subsites –1 and +1, the two sugar rings being twisted to become almost perpendicular to each other. Catalytically important residues are rather concentrated around this region. Glu70 (Glu78/Glu94 in pXyl and CelA) has been identified as the proton donor (1Honda Y. Kitaoka M. J. Biol. Chem. 2004; 279: 55097-55103Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The catalytic base residue (Asp263/Asp281/Asp278 in Rex, pXyl, and CelA) holds a water molecule through hydrogen bonds together with a conserved Tyr residue (Tyr198/Tyr203/Tyr215). The water is thought to correspond to the nucleophilic water, which is activated by the catalytic base residue (11Guérin D.M. Lascombe M.B. Costabel M. Souchon H. Lamzin V. Béguin P. Alzari P.M. J. Mol. Biol. 2002; 316: 1061-1069Crossref PubMed Scopus (121) Google Scholar). In the CelA-cellopentaose complex structure, the third catalytically important residue (Asp128/Asp144/Asp152) forms bifurcated hydrogen bonds with the O-2 and O-3 atoms of the glucose unit at subsite –1 with the 2,5B conformation (11Guérin D.M. Lascombe M.B. Costabel M. Souchon H. Lamzin V. Béguin P. Alzari P.M. J. Mol. Biol. 2002; 316: 1061-1069Crossref PubMed Scopus (121) Google Scholar). The interaction is critical to stabilize the sugar ring in a strained boat conformation, which is thought to be a prerequisite for the inverting hydrolytic mechanism through a transition state with the oxocarbenium ion, the planarity of the atoms C-5, O-5, C-1, and C-2 facilitating the formation of a partial double bond between O-5 and C-1 (11Guérin D.M. Lascombe M.B. Costabel M. Souchon H. Lamzin V. Béguin P. Alzari P.M. J. Mol. Biol. 2002; 316: 1061-1069Crossref PubMed Scopus (121) Google Scholar). In the E70A-xylobiose structure, Asp128 does not form any interaction with the ligand. However, the side chain of Asp128 is positioned similarly with that of Asp152 of CelA, and it would also stabilize the xylose ring at subsite –1 if the ring takes on a boat conformation. In summary, the catalytic mechanism of Rex seems to be basically conserved with other GH-8a enzymes.At subsite +1, the xylose molecule in Rex overlaps better with a glucose unit of the product cellotriose molecule in CelA, rather than that of the uncleaved cellopentaose molecule. For CelA, the product molecule at subsites +1 to +3 is thought to represent a possible first step during which the leaving group rotates slightly and shifts away from the reaction center, the stacking interactions being preserved (11Guérin D.M. Lascombe M.B. Costabel M. Souchon H. Lamzin V. Béguin P. Alzari P.M. J. Mol. Biol. 2002; 316: 1061-1069Crossref PubMed Scopus (121) Google Scholar). The xylose molecule in WT-xylose of Rex also seems to correspond to a product molecule being released. Subsite +1 of Rex is unique compared with that of other GH-8a enzymes, because the residues at this subsite (Asp61, Asn64, Arg68, Ser262, His319, and Arg357) are not conserved in CelA and pXyl. However, the stacking Tyr residue (Tyr360/Tyr381/ Tyr372 in Rex, pXyl, and CelA) is conserved and fixes the sugar unit at an approximate position.The most notable difference between Rex and the other two GH-8a enzymes is the blockage of subsite +2 by the kink in the loop before α10 at Ser317-Pro320 (Fig. 4b). His319 is directly hydrogen-bonded with the β-hydroxyl of the xylose at subsite +1, contributing to the discrimination of the anomers at the reducing end. Leu318 blocks subsite +2 with its long side chain together with His319. A proline residue (Pro320) is present only in Rex, and the main chain trace bends at an almost right angle at this position. The bent loop structure seems to be intrinsically stable, because there is no conformational difference between the unliganded and liganded structures. On the other hand, in pXyl and CelA, this loop is located away from the substrate. Instead, a Tyr residue in the loop before α12 (Tyr378/Tyr369 in pXyl and CelA) forms subsite +3.Specificity for Xylosides—The order of preference of Rex for xylo-/glucooligosaccharides is XXX ≫ GXX > XXG ≫ GXG (where G represents glucose and X is xylose), and no detectable activity is observed for XGG, GGX, and XGX (1Honda Y. Kitaoka M. J. Biol. Chem. 2004; 279: 55097-55103Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). This indicates that subsite –1 strictly requires a xyloside, and the other two subsites also exhibit a c" @default.
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• W2065905475 title "Structural Basis for the Specificity of the Reducing End Xylose-releasing Exo-oligoxylanase from Bacillus halodurans C-125" @default.
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