Structure and Function of a Family 10 β-Xylanase Chimera of Streptomyces olivaceoviridis E-86 FXYN and Cellulomonas fimi Cex

The catalytic domain of xylanases belonging to glycoside hydrolase family 10 (GH10) can be divided into 22 modules (M1 to M22; Sato, Y., Niimura, Y., Yura, K., and Go, M. (1999) Gene 238, 93-101). Inspection of the crystal structure of a GH10 xylanase from Streptomyces olivaceoviridis E-86 (SoXyn10A) revealed that the catalytic domain of GH10 xylanases can be dissected into two parts, an N-terminal larger region and C-terminal smaller region, by the substrate binding cleft, corresponding to the module border between M14 and M15. It has been suggested that the topology of the substrate binding clefts of GH10 xylanases are not conserved (Charnock, S.J., Spurway, T.D., Xie, H., Beylot, M.H., Virden, R., Warren, R.A.J., Hazlewood, G.P., and Gilbert, H.J. (1998) J. Biol. Chem. 273, 32187-32199). To facilitate a greater understanding of the structure-function relationship of the substrate binding cleft of GH10 xylanases, a chimeric xylanase between SoXyn10A and Xyn10A from Cellulomonas fimi (CfXyn10A) was constructed and the topology of the hybrid substrate binding cleft established. At the three-dimensional level, SoXyn10A and CfXyn10A appear to possess 5 subsites, with the amino acid residues comprising subsites –3 to +1 being well conserved, while the +2 subsites are quite different. Biochemical analyses of the chimeric enzyme along with SoXyn10A and CfXyn10A indicated that differences in the structure of subsite +2 influence bond cleavage frequencies and the catalytic efficiency of xylooligosaccharide hydrolysis. The hybrid enzyme constructed in this study displays fascinating biochemistry, with an interesting combination of properties from the parent enzymes, resulting in a low production of xylose. 3 by gest on Sptem er 1, 2017 hp://w w w .jb.org/ D ow nladed from INTRODUCTION The plant cell wall consists mainly of a complex mixture of polysaccharides such as cellulose, pectins and hemicellulose (1), where the latter is comprised mainly of xylan. The backbone of xylan is formed by β-1,4-linked D-xylopyranose units to which several side groups such as α-1,2-linked 4-O-methyl D-glucuronic acid and α-1,3-linked L-arabinofuranose are attached (2). β-Xylanases (EC 3.2.1.8) randomly hydrolyze β-1,4-glycosidic linkages within the xylan backbone via a double displacement mechanism involving a carboxylate functioning as a proton donor (the acid/base catalyst), which facilitates the dissociation of the poor glycosidic leaving group, and a nucleophilic carboxylate involved in formation of a covalently linked enzyme-substrate intermediate (3). On the basis of primary structure homology, the majority of xylanases have been classified into glycoside hydrolase families 10 and 11 (GH10 and GH11, respectively) (4). The three-dimensional (3D) structures of ten GH10 xylanases have now been solved (5-11). They all have very similar structures, comprising (β/α)8-barrels as well as additional helices and loops which are arranged in a basic TIM-barrel structure forming the active site cleft (9). The cleft forms deep grooves consistent with the endo-mode of action, and comprises a series of subsites, each one tailored towards the binding of a single xylose moiety (11). The subsites that bind the glycone and aglycone regions of the substrate are prefixed by (-) and (+), respectively, and their numbers are related to the proximity to the site of bond cleavage (the glycosidic bond between the +1 and -1 subsites is cleaved by the enzyme (12)). Analysis of active site amino acids playing important roles in substrate binding and catalysis have been extensively facilitated by the elucidation of crystal structures of GH10 xylanases covalently linked to mechanism-based cellobiosyl and xylobiosyl inhibitors (13) and also by site directed mutagenesis (14, 15). Although GH10 xylanases display the same 4 by gest on Sptem er 1, 2017 hp://w w w .jb.org/ D ow nladed from gross fold, the topology of their substrate binding clefts is not conserved (15), and thus differences in substrate specificity could be expected. Cellulomonas fimi endo-xylanase Xyn10A (CfXyn10A, formerly known as Cex) is one of the most characterized xylanases (13, 16-22). Based on the 3D structure of CfXyn10A, the catalytic domain has been divided into 22 modules (M1 to M22) (23), where a module is defined as a contiguous polypeptide segment of a protein which has a compact conformation within a globular domain. A module is defined by the distance between Cα atoms, and on average a module is about 15 amino acid residues long (24). Interestingly, thirty-one intron sites in fungal genes encoding GH10 xylanases were found to correlate to module boundaries with considerable statistical significance (23). This indicates that the location of introns in eukaryotic xylanase genes are not random and supports the concept that introns play an important role in protein evolution as mediators of exon shuffling (25). Therefore, module shuffling in vitro mimics one of the natural mechanisms of protein evolution. Thus, module shuffling was selected as a tool to study the structure-function relationships of xylanases. The GH10 enzyme Xyn10A from Streptomyces olivaceoviridis E-86 (SoXyn10A, formerly known as FXYN) was selected as one of the parent enzymes as this enzyme is amenable to crystallization (11, 26), and has been cloned (27). In addition, the substrate specificity of SoXyn10A has been well characterized (28-36). Further, we have succeeded in soaking xylotriose into both the glycon and aglycon region of the substrate binding cleft (37). As shown in Figure 1, the catalytic domain of GH10 xylanases is sub-divided into two parts which consist of the N-terminal larger region and the C-terminal smaller region. The boundary between module M14 and M15 corresponds to the border between these two regions. In the present investigation, we selected the boundary between modules M14 and M15 for shuffling to understand the 5 by gest on Sptem er 1, 2017 hp://w w w .jb.org/ D ow nladed from topology and function of the substrate binding cleft. In this paper, the structure and function of the resultant chimeric GH10 xylanases are described. MATERIALS AND METHODS Construction of chimeric enzymes—The catalytic domain of SoXyn10A and CfXyn10A were separately subcloned into the pQE60 vector (QIAGEN, Hilden, Germany). Construction of the chimera was performed by the polymerase chain reaction (PCR) using overlapping primers at their respective module boundaries (Figure 2). The DNA fragment from SoXyn10A encoding modules M1 to M14 and the DNA fragment from CfXyn10A encoding modules M15 to M22 were amplified using the following sets of primers for FC-14-15 (M1-M14 sense: 5’-CCA TGG GCT CCT ACG CCC TTC CCA GAT CAG-3’; M1-M14 antisense: 5’-GCG ACT GGA AGC CAA CGC AGT CGA TTG GCA CGC C-3’; M15-M22 sense; 5’-CTG CGT CGG CTT CCA GTC GCA CCT CAT CGT CGG CC-3’; M15-M22 antisense: 5’-GGA TCC GAA GGC TTC CAT CAC GGC GGC GTA GG-3’) and for CF-14-15 (1-14 sense: 5’CCA TGG CTA GGA CCA CGC CCG CAC CCG -3’; M1-M14 antisense: 5’-GCG ACT GGA ATC CTA CAC AGT CGA GCG GGA CGC C-3’; M15-M22 sense; 5’-CTG CGT CGG GTT CCA GTC ACA CTT CAA CAG CGG CAG C-3’; M15-M22 antisense: 5’GGA TCC AGC GTT GAG GAC GGC GGT GTA GGC AGC -3’). Each of the 25 amplification cycles consisted of denaturation at 98C for 1 min and annealing and primer extension at 72C for 1 min. The 10 bp overlapping regions (underlined) of the primers were designed to be complementary at their respective module boundaries. The first round of PCR products were separated by agarose gel electrophoresis, followed by gel extraction, and used for the second round PCR without primer. Each of the 20 amplification cycles consisted of denaturation at 98C for 1 min, annealing at 60C for 6 by gest on Sptem er 1, 2017 hp://w w w .jb.org/ D ow nladed from 25 min and primer extension at 72C for 5 min. The strands having matching sequences at their respective module boundaries overlapped and acted as primers for each other. On the 3rd round of PCR, the combined fragment was amplified with PCR primers for FC-14-15 (sense: 5’-CCA TGG GCT CCT ACG CCC TTC CCA GAT CAG-3’ and antisense: 5’-GGA TCC GAA GGC TTC CAT CAC GGC GGC GTA GG-3’) for CF-14-15 (sense: 5’-CCA TGG CTA GGA CCA CGC CCG CAC CCG-3’ and antisense: 5’-GGA TCC AGC GTT GAG GAC GGC GGT GTA GGC AGC-3’) by 25 cycle of shuttle PCR with denaturation at 98C for 1 min and annealing and primer extension at 72C for 1 min. Gene Expression and Protein Purification—For expression in E. coli and purification of the SoXyn10A, CfXyn10A, FC-14-15 and CF-14-15, the pET expression system (NOVAGEN, Madison, WI, USA) was employed. Thus, each gene was individually inserted into the pET28 vector (to yield pETfxyn, pETcex, pETfc-14-15, and pETcf-14-15, respectively). The enzymes were expressed as fusion proteins that comprised each enzyme plus a carboxyl-terminal tag of six histidine residues. The recombinant plasmids were used to transform E. coli BL21 (DE3) and transformants were cultivated at 25°C in LB medium (1 liter) that contained kanamycin (20 μg/ml) until the optical density at 600 nm reached 0.4. After addition of isopropyl-1-thio-β-D-galactoside (IPTG) to a final concentration of 1 mM, the culture was incubated at 25C for 24 h. After the E. coli cells were removed from the culture by centrifugation (6,000 x g, 10 min), ammonium sulphate was added to give a 70% saturation level and the resulting mixture was kept at 4C for 16 h. The precipitate was collected by centrifugation (10,000 x g, 20 min) and dissolved in a small amount of distilled water followed by dialysis against deionized water. After removal of insoluble 7 by gest on Sptem er 1, 2017 hp://w w w .jb.org/ D ow nladed from material by centrifugation (12,000 g, 30 min), the obtained solution was then loaded on a HisTrapTM chelating column (Amersham Bioscience, Piscataway, NJ, USA). The bound enzyme was eluted with a 50 mM phosphate buffer (pH 7.0) containing 250 mM imidazole. The elution of the enzyme was monitored by sodium dodecylsulfate-polyacryamide gel electrophoresis (SDS-PAGE) (38). The enzyme eluted as a homogenous protein as detected by SDS-PAGE, and the relevant fractions were pooled and dialyzed against deionized water. Crystallization and data collection—Crystallization trials of FC-14-15 were conducted using the modified crystallization conditions for SoXyn10A (26). FC-14-15 was crystallized by the hanging drop vapor diffusion method at room temperature using a 20 mg/ml protein solution and a reservoir solution comprising 1.2 M ammonium sulfate and 2% McIlvaine buffer (a mixture of 0.1 M citric acid and 0.2 M Na2HPO4, pH 5.5). After two weeks, thin needle crystal clusters grew to a size of 0.1 x 0.1 x 0.4 mm. Diffraction experiments were conducted at beam-line 6A at the Photon Factory in Tsukuba, Japan, with a Weissenberg camera for macromolecules (39) at room temperature. The collimator used was 0.1 mm and the wavelength was 1.0 Å. Intensity data covering a total rotation of 180° was collected using a single crystal. Diffraction data were detected using a imaging plate (20 x 40 cm) and digitized by BA2000 (Fuji film, Tokyo, Japan). Crystals of FC-14-15 diffracted beyond 2.0 Å resolution. The data sets were processed using the program DENZO, and scaled using the program SCALEPACK (40). The crystals are orthorhombic and belong to space group P212121, with cell dimensions of a = 48.48Å, b = 57.19Å, and c = 106.67Å, containing one molecule in the asymmetric unit. The collected native intensity data set included a total 8 by gest on Sptem er 1, 2017 hp://w w w .jb.org/ D ow nladed from of 52,375 observations, which were reduced to 13,853 unique reflections with a completeness of 88.3% and a merging R-factor of 0.065 against the data from 100 to 2.2 Å resolution. Structure determination—The structural analysis was performed using the molecular replacement method with the program AMoRe (41) in the CCP4 suite (42) (Table 1). Two structures of SoXyn10A (PDB accession code 1xyf) and CfXyn10A (2exo) were superimposed based on topological alignment. A 3D model of FC-14-15 was built by connecting the C-terminal 1-203 residues of SoXyn10A and C-terminal 202-310 of CfXyn10A and was used as a search model. This model was subjected to a rotation search using the 8.0-3.5 Å resolution data. A single high peak had correlation coefficients of 0.189 and the following translation search resulted in a correlation coefficient of 0.510 and an R-value of 41.3%. The model thus positioned was subjected to cycles of rigid-body refinement against the data from 30 to 2.2 Å resolution using the program CNS (43), resulting in proper structure models. Ten percent of the observed reflections were randomly removed for cross-validation (44). The structure was refined by iterative cycles of simulated annealing and manual model rebuilding, conducted on the resultant 2Fobs-Fcalc and Fobs-Fcalc maps, using the program CNS. The structure was refined to an R-factor of 15.3 % and an Rfree-factor of 19.8%. The stereochemistry of the model was analyzed with the program PROCHECK (45). In a Ramachandran plot (46), 91.7 % of the non-glycine residues were in the most favoured regions of phi-psi plot and the remainder were in additional allowed regions. Enzyme Assays and Source of the Substrate—Steady-state kinetics were investigated as previously reported (33). Briefly, the reaction mixture containing 250 μl 9 by gest on Sptem er 1, 2017 hp://w w w .jb.org/ D ow nladed from of substrate solution (p-nitrophenyl-β-D-xylobioside (PNP-X2) or p-nitrophenyl-β-D-cellobioside (PNP-G2)) at various concentrations, 150 μl of McIlvaine buffer (a mixture of 0.1 M citric acid and 0.2 M Na2HPO4, pH 7.0) and 50 μl of 1% (w/v) BSA (bovine serum albumin) was incubated at 30C for 5 min and then 50 μl of enzyme solution was added. The amount of p-nitrophenol released was determined by monitoring the absorbance at 400 nm as a function of time with a spectrometer (DU-7400; Beckman, Palo Alto, CA). The kinetic parameters kcat and Km were determined by Eadie-Hofstee plot from three independent experiments, and at a five substrate concentrations. PNP-X2 was synthesized by the method described in a previous paper (47). The xylobiose used in the synthesis was purified from ‘Xylobiose Mixture’ (SUNTORY Ltd., Osaka, Japan). PNP-G2 was a generous gift from Yaizu Suisan Co. Ltd. (Yaizu, Japan). For the hydrolysis of soluble birchwood xylan, reaction mixtures containing 150 μl of MacIlvaine buffer, 50 μl of 1% BSA (w/v) and 250 μl of birchwood xylan solution (0.2 mg/ml; Fulka, Neu-Ulm, Swizerland) were equilibrated at 30C for 5 min, and then reactions were initiated by the addition of 50 μl of enzyme solution (the final concentrations of SoXyn10A, CfXyn10A and FC-14-15 were 0.002, 0.004 and 0.005 mg/ml). The increase in reducing power was measured by the method of Fox and Robyt (48). The hydrolysis products were also analyzed using a Carbo Pac PA-1 column (Dionex Co. Ltd., Sunnyvale, CA, USA) with high performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) system with a flow rate of 1 ml/min and elution with 0.1 M NaOH (0-5 min), followed by a linear gradient (5-35 min) of sodium acetate (0-0.4 M). Substituted xylooligosaccharides such as 4-O-methyl-α-D-glucuronosyl(1 2)-β-D-xylopyranosyl-(1 4)-β-D-xylopyranosyl-(1 4)-β-D-xylopyranosyl-(1 4)-β 10 by gest on Sptem er 1, 2017 hp://w w w .jb.org/ D ow nladed from -D-xylopyranoside (4MeGlcUAX4) and 4-O-methyl-α-D-glucuronosyl-(1 2)-β-Dxylopyranosyl-(1 4)-β-D-xylopyranosyl-(1 4)-β-D-xylopyranoside (4MeGlcUAX3) were prepared from the final reaction products of birchwood xylan hydrolysis by FC-14-15 according to the method described previously (30) and the structure of the oligosaccharides determined by nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometory (ESI-MS). H and C NMR experiments were recorded at 303 K with a Bruker Avance-500 spectrometer. ESI-MS was performed in the negative ion mode. Solutions of oligosaccharides (100 μg) in aqueous 30% methanol containing 0.75% HCl (100 μl) were infused into the electrospray source at 4 μl per min. The ion spray was operated at 5000 V with an orifice potential of 35 V. Ten scans (100-2500 amu) were collected and averaged. Bond cleavage frequencies (BCFs) and activities for the hydrolysis of xylooligosaccharides, and the calculation of subsite binding energies were performed as described previously (15). These values were determined from three independent experiments. To evaluate the catalytic efficiencies of the xylanases against xylooligosaccharides, 0.3–800 nM of enzyme were incubated with 10 μM of substrate in McIlvaine buffer, pH 7.0 for up to 200 min at 30°C. At regular time intervals, a 0.1-ml aliquot was removed, the enzyme was inactivated by adding sodium hydroxide to the concentration of 0.1 M, and the xylooligosaccharides in the samples were quantified by HPAEC-PAD as described above using L-fucose as an internal standard. The progress curves of oligosaccharide cleavage were used to determine the kcat/Km of the reaction using the following equation described by Matsui et al. (49, 50), k . t = ln([S0] / [St]) (Eq. 1) 11 by gest on Sptem er 1, 2017 hp://w w w .jb.org/ D ow nladed from where k = (kcat/Km)[E], t represents time, and [S0] and [St] represent substrate concentrations at time 0 and t, respectively. This relationship is only valid when [E] << [S] << Km. The concentration of substrate in these reactions was < CfXyn10A > FC-14-15. The hydrolysis rates of the substrate by FC-14-15 and CfXyn10A were similar, whereas SoXyn10A hydrolyzed 4MeGlcUAX4 with a significantly higher extent than the other enzymes. This can be explained from the result of BCF (Figure 5) and the structures of complexes with substituted oligosaccharides (54). According to the xylotriose-bound structure of SoXyn10A, the O-2 atoms of bound xylose at subsite -1 and -2 were buried into the cleft and could not have attached branches. Therefore, to hydrolyze 4MeGlcUAX4, the enzymes have to cleave the first linkage from reducing end to produce xylose and 4MeGlcUAX3. From the BCF (Figure 5), it can be seen that SoXyn10A is able to hydrolyze the first linkage from reducing end but the other enzymes cannot, explaining why CfXyn10A and FC-14-15 are not good at hydrolyzing 4MeGlcUAX4. In the case of CfXyn10A and FC-14-15, the production of xylose was dependent on the hydrolysis of xylotriose and 4MeGlcUAX4 since xylose was not produced by the degradation of xylooligosaccharides longer than xylotriose (Figure 5). In contrast, SoXyn10A produced xylose when the enzyme cleaved xylotetraose in addition to the above substrates (Figure 5). CfXyn10A hydrolyzes xylotriose more efficiently than SoXyn10A and FC-14-15 (Figure 4), while SoXyn10A hydrolyzes 4MeGlcUAX4 more efficient than CfXyn10A and FC-14-15 (Figure 8). Since FC-14-15 had the weakest reactivity for both substrates, a lower production of xylose was observed than with SoXyn10A and CfXyn10A. Conclusion— The results of this study suggest that the topology of the 19 by gest on Sptem er 1, 2017 hp://w w w .jb.org/ D ow nladed from substrate binding cleft is determined by the environment of the glycon side especially in subsite +2 which is not conserved in GH10 xylanases. The hybrid enzyme that we constructed in this study is fascinating, with an interesting combination of properties from the parent enzymes, resulting in low production of xylose. It is already known that the structures of the glycon side of the active site in GH10 xylanases are highly conserved however in the aglycon side the structures are very different. The effects of these differences on substrate specificity have not previously been elucidated, in spite of the topology of the substrate binding cleft of GH10 xylanases not being conserved. The structure of subsites -3 to +1 of the substrate binding clefts of SoXyn10A and CfXyn10A were not significantly different, however differences in the topology of the clefts between the two enzymes could be found. Especially, these differences were remarkable when the enzymes used subsite +2. Not only the properties of hydrolysis of linear xylooligosaccharides, but also of hydrolysis of substituted oligosaccharides were affected. Acknowledgments—The authors are grateful to Dr. Simon J. Charnock for the useful comments on analysis of BCFs. The authors are also grateful to SUNTORY Ltd. for supplying the xylobiose mixture. We wish to thank Dr. Leila Lo Leggio for valuable discussions and critically reading this manuscript. This work was supported in part by Grants for the Program for Promotion of Basic Research Activities for InnovativeBiosciences and Grant-in-Aid for Scientific Research. REFERENCES1. Carpita, N. C., and Gibeaut, D. M. (1993) Plant J. 3, 1-302. Timell, T. E. 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Chem., 279, 9606-9614 24bygestonSptemer1,2017hp://www.jb.org/Downladedfrom Table 1 X-ray diffraction data and refinement statistics for FC-14-15______________________________________________________________________Cell parameters (P212121)a (Å)48.48b (Å)57.19c (Å)106.67Resolution range (Å)30-2.2(2.34-2.20) No. of reflections in refinement137141178Completeness (%)88.3(78.1)R-factor (%)15.3(16.2)Rfree-factor (%)19.8(21.6) No. of protein non-hydrogen atoms 2458No. of water molecules240 rmsdBond lengths (Å)0.005Bond angles (deg.)1.3Dihedral angles (deg.)21.5 ______________________________________________________________________ 25bygestonSptemer1,2017hp://www.jb.org/Downladedfrom Table 2 Kinetic parameters of chimeric xylanasePNP-G2PNP-X2Km (mM) kcat (s) kcat/Km(s/mM)Km (mM) kcat (s) kcat/Km(s/mM)SoXyn10A 97±5 2.2±0.2 0.023±0.003 2.0±0.1 46±2 23±2CfXyn10A 0.71±0.04 2.5±0.1 3.6±0.4 0.013±0.001 8.6±0.2 660±70FC-14-15 13±1 3.4±0.8 0.26±0.09 0.22±0.01 74±1 340±20 26bygestonSptemer1,2017hp://www.jb.org/Downladedfrom Figure 1 Structure of SoXyn10A, CfXyn10A and FC-14-15A: SoXyn10A (RCSB Protein Data Bank accession number 1ISX), B: FC-14-15, C:CfXyn10A (RCSB Protein Data Bank accession number 2XYL). The crystal analysis of the FC-14-15 were performed at 2.2Å resolution. The coordinate for FC-14-15 havebeen deposited in the RCSB Protein Data Bank (accession number 1v6y). The crystalstructure of GH10 xylanases indicate that the catalytic domain of the enzymes aresubdivided into two parts separated by the module boundary between module M14 and M15 (modules M1-M14, purple; modules M15-M22, yellow). The boundary betweenM14 and M15 correspond with the substrate binding cleft of GH10 xylanases. Figure 2 Construction of FC-14-15A: Schematic representation of the boundary of module M1-M22 and the constructedchimeric enzymes. B: The chimeric xylanases were constructed by three times PCR with overlapping primers. Figure 3 Substrate binding cleft of SoXyn10A, CfXyn10A, FC-14-15Superposition of the substrate binding cleft of FC-14-15 (green), SoXyn10A (modules from M15 to M22; pink) and CfXyn10A (modules from M1 to M14; orange).Xylotriose inserted into the SoXyn10A cleft were modeled from the SoXyn10A/xylotriose complex structure (37). The numbering of amino acid residues inblack referred to the amino acid number of SoXyn10A and the numbering in orangereferred to the amino acid number of CfXyn10A. Figure 4 Rate of xylooligosaccharide hydrolysis by CfXyn10A, SoXyn10A andFC-14-15 27bygestonSptemer1,2017hp://www.jb.org/Downladedfrom SoXyn10A (), CfXyn10A (), and FC-14-15 () wereincubated with xylooligosaccharides of different lengths and the rate of substratehydrolysis was used to calculate kcat/Km. Figure 5 Bond cleavage frequencies of xylooligosaccharides by CfXyn10A,SoXyn10A, and FC-14-15 Figure 6 Binding energies of the subsites of the substrate binding sites ofCfXyn10A, SoXyn10A and FC-14-15The binding energies of the subsites of SoXyn10A (A), CfXyn10A (B), and FC-14-15(C) were calculated using the method described previously (15), substituting thekcat/Kmdata displayed in Figure 4 and the BCFs exhibited in Figure 5. Figure 7 HPAEC-PAD analysis of soluble birchwood xylan hydrolysis byCfXyn10A, SoXyn10A and FC-14-15Birchwood xylan hydrolysate by SoXyn10A (A), CfXyn10A (B), and FC-14-15 (C)were applied to HPAEC-PAD system. The positions at which xylose (a), xylobiose (b),xylotriose (c), xylotetraose (d), and xylooligosaccharides substituted by 4-O-methylglucuronic acid (e) were eluted from the HPAEC column are indicated. Figure 8 Hydrolysis of 4MeGlcUAX4 by CfXyn10A, SoXyn10A and FC-14-15SoXyn10A (), CfXyn10A (), and FC-14-15 () wereincubated with 4MeGlcUAX4, and the extent of substrate hydrolysis was detected by