Construction of Thermophilic Xylanase and Its Structural Analysis

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Construction of thermophilic xylanase and its structural analysis Masahiro Watanabe, Harumi Fukada, and Kazuhiko Ishikawa Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00414 • Publication Date (Web): 13 Jul 2016 Downloaded from http://pubs.acs.org on July 29, 2016

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Biochemistry

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Construction of thermophilic xylanase and its structural analysis

2 Masahiro Watanabe1,2, Harumi Fukada3 and Kazuhiko Ishikawa1,4

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1

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Technology (AIST), 3-11-32 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046, Japan

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2

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Science and Technology (AIST), 3-11-32 Kagamiyama, Higashi-Hiroshima, Hiroshima

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739-0046, Japan

Biomass Refinery Research Center, National Institute of Advanced Industrial Science and

Research Institute for Sustainable Chemistry, National Institute of Advanced Industrial

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Gakuen-cho, Nakaku, Sakai, Osaka 599-8531, Japan

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Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka, 563-8577, Japan

Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1

Biomedical Research Institute, National Institute of Advanced Industrial Science and

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*Running title: Construction of thermophilic xylanase

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To whom correspondence should be addressed: 3-11-32 Kagamiyama, Higashi-Hiroshima,

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Hiroshima 739-0046, Japan. Tel.: +81-82-420-8258, Fax: +81-82-420-7820, E-mail:

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[email protected]

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Keywords: biomass; cellulase; hemi-cellulase; protein engineering; thermostability; X-ray

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crystallography; differential scanning calorimetry; fungus

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Background: Thermophilic xylanase holds a great deal of potential in many industrial

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applications for biomass reduction.

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Results: By modifying the N-terminal region of a xylanase, thermophilic mutants with a high

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melting temperature were constructed, and the structure of the mutant was analyzed.

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Conclusion: By increasing C-C contacts, stabilizing loop structure, and introducing disulfide

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bond in the N-terminal region, the optimum temperature and thermostability of the xylanase

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was improved.

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Significance: The thermostability of a xylanase was improved by over 20°C by modifying the

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N-terminal region.

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ABSTRACT

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The Glycoside hydrolase family 11 xylanase has been utilized in a wide variety of industrial

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applications, from food processing to kraft pulp bleaching. Thermostability enhances the

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economic value of industrial enzymes by making them more robust. Recently we solved the

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crystal structure of an endo-ß-1,4-xylanase (GH11) from mesophilic Talaromyces

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cellulolyticus, named XylC. Ligand-free XylC exists to two conformations (open/closed

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forms). We found that the “closed” structure possessed an unstable region within the

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N-terminal region far from the active site. In this study, we designed the thermostable xylanase

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by the structure-based site-directed mutagenesis on the N-terminal region. In total nine

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mutations (S35C, N44H, Y61M, T62C, N63L, D65P, N66G, T101P, and S102N) and an

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introduced disulfide bond of the enzyme were contributed to the improvement in

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thermostability. By combining the mutations, we succeeded in constructing a mutant of which

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the melting temperature was partially additively increased by over 20°C (measured by a 2 ACS Paragon Plus Environment

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Biochemistry

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differential scanning calorimetry) and the activity was additively enhanced at elevated

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temperatures, without loss of the original specific activity. The crystal structure of the most

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thermostable mutant was determined at 2.0-Å resolution to elucidate the structural basis of

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thermostability. From the crystal structure of the mutant, it was revealed that the formation of a

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disulfide bond induces new C-C contacts and a conformational change in the N-terminus. The

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resulting induced conformational change in the N-terminus is key for stabilizing this region and

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for constructing thermostable mutants without compromising the activity.

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INTRODUCTION

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Cellulosic materials constitute most of the biomass on earth and can be converted into

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biofuels or bio-based materials. Such conversions utilize fermentable sugars obtained from

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cellulosic biomass by the activity of cellulase-related enzymes (1-3). Xylan is a major structural

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component of the plant cell wall and is the second most abundant renewable biomass resource

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(4, 5). Xylan consists of a ß-1,4-D-xylan backbone with short side chains of O-acetyl,

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ß-L-arabinofuranosyl, D-beta-glucuronic acid, and phenolic acid (6). Cellulose and xylan are

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closely linked together in plant cell walls (7). Thus, cellulases and hemicellulases work

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coordinately in the enzymatic degradation of these polysaccharides. Filamentous fungi produce

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a wide spectrum of enzymes for degrading cellulose and xylan (8). Xylanases (endo-

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ß-1,4-xylanases; EC 3.2.1.8) catalyze the hydrolysis of the ß-1,4 bonds of xylan and are thus

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important enzymes for the degradation of hemicellulosic polysaccharides in lignocellulosic

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biomass (9, 10). Based on their amino acid sequence similarities, xylanases are classified 3 ACS Paragon Plus Environment

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primarily into glycoside hydrolase (GH; http://www.cazy.org/Glycoside-Hydrolases.html)

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families 10 and 11 (11). GH10 xylanases generally have a molecular weight ≥30 kDa with a

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low pI value, whereas GH11 xylanases are generally smaller (approximately 20 kDa) with a

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high pI value (12). The crystal structures of xylanases show that GH10 enzymes fold into a

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(ß/α)8-barrel (13-15), whereas GH11 enzymes have a ß-jelly roll structure (16-18).

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Talaromyces cellulolyticus (19), which was isolated by Yamanobe et al. (20),

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produces an abundance of cellulolytic enzymes. Fujii et al. (21) reported that T. cellulolyticus

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culture supernatant has a higher cellulase specific activity and yields more glucose from

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lignocellulosic materials than does Trichoderma reesei (T. reesei) culture supernatant. Six of

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the seven GH11 xylanases produced by T. cellulolyticus were recently cloned and characterized

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by Watanabe et al. (22). Of the six xylanases characterized, TcXylC had the highest activity

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against xylan, despite the low expression level of the gene (22). In addition, a system for the

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over-expression and purification of recombinant TcXylC using E.coli was developed and the

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crystal structure of TcXylC was solved at 1.98-Å resolution (23). Structural analysis revealed

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that TcXylC consists of a common ß-jelly roll structure exhibiting a two-state structure (open-

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and closed-form) in the asymmetric unit. RMSD of the Cα atoms between the open and closed

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forms was 0.55 Å. However, the only closed form possesses a tunnel structure of the active site

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formed by Trp52 and Pro159 (23). The tunnel structure retained hydrogen-bonding networks

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with 10 water molecules and is thought to mimic the structure of the closed form. The

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N-terminal region including Trp52 in the closed form exhibits high B-factor values (23).

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Conformational changes in the flexible region are presumed to induce and accelerate the 4 ACS Paragon Plus Environment

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enzymatic reaction. Although the structures and functions of GH11 xylanases from many

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species have been well characterized (24-26) and compared (14, 16), the structural basis of

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xylanase thermostability remains unclear, due in part to the challenge of selecting an

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appropriate residue (or location) for site-directed mutagenesis, as even minimal substitution of

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residues can lead to misfolding of the target protein (27-29). Some disulfide bond designs

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tested increased the irreversible thermostability of the B. circulans xylanase, but not all

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enhanced the activity of the enzyme at elevated temperatures (30).

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TcXylC exhibits the highest activity of the xylanases produced by T. cellulolyticus

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and has been expected to be used for industrial application. In this study, therefore, we

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engineered TcXylC to improve both its thermostability and optimum temperature using

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site-directed mutagenesis and elucidated the structural basis for thermostabilization.

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EXPERIMENTAL PROCEDURES

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Cloning of TcXylC and its variants.

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The genes encoding TcXylC without the putative signal peptide (34 amino acid

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residues) and its variants were cloned into pET11a (Novagen) at the NdeI and BamHI

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restriction enzyme sites, such that the NdeI site (CATATG) included the initiator methionine

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codon (23). All mutant genes were constructed by PCR using a PrimeSTAR Max DNA

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Polymerase kit (Takara Bio) and the primers listed in Supplemental Table 1.

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Protein expression and purification. 5 ACS Paragon Plus Environment

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Plasmids encoding TcXylC and its mutants were transformed into E. coli BL21

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(DE3) cells (Novagen). Cells were cultured at 37°C in Luria-Bertani medium containing 100

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mg/L of ampicillin-Na. When the culture reached an OD600 of 0.6, expression of recombinant

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protein was induced by addition of isopropyl ß-D-1-thiogalactopyranoside (IPTG) to a final

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concentration of 200 µM. The culture was subsequently incubated at 20°C for 16 h, after

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which the cells were harvested by centrifugation at 5,000 × g for 15 min at 4°C. The resulting

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cell pellets were resuspended in 20 mM Tris-HCl (pH 8.0) containing 50 mM NaCl. Soluble

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protein was extracted by three freeze-thaw cycles (freezing at −80°C for 1 h followed by

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complete thawing at room temperature) and recovered by centrifugation at 35,870 × g for 20

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min at 4°C. The recombinant proteins were purified using a modified method described

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previously (23). The supernatants were applied to a Q-HP column (GE Healthcare)

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equilibrated in 20 mM Tris-HCl (pH 8.0) and eluted with an ascending salt concentration

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gradient. Wild-type (WT) and mutant TcXylC proteins were eluted from the column at

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between 200 and 400 mM NaCl. Fractions containing the recombinant protein were collected,

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dialyzed against 20 mM Tris-HCl (pH 8.0)/2 M NaCl, and applied to a Butyl HP column (GE

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Healthcare) equilibrated in the same buffer. Proteins were eluted with an ascending salt

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concentration gradient. The recombinant protein was found to elute at approximately 1 M

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NaCl. As a final step, the eluted protein was applied directly to a Superdex 200 16/60 gel

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filtration column (GE Healthcare) equilibrated with 20 mM Tris-HCl (pH 8.0)/50 mM NaCl.

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The purity of the target protein was assessed using SDS-PAGE, and the xylanase activity was


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Biochemistry

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also determined (Supplemental Fig. 1). All TcXylC mutants were purified using a method

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similar to that used for the WT protein.

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Protein assay.

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All protein concentrations were determined according to the BCA method (31),

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with bovine serum albumin as a standard. Protein solution (25 µL) was mixed with 200 µL of

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BCA reagent (Thermo Scientific Pierce) and then incubated at 37°C for 30 min, after which

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the concentration was determined by measuring the absorption at 570 nm in comparison with

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standards of known concentration.

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Enzyme assay.

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Xylanase activity was measured as reported previously (32) using birch-wood xylan

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as the substrate under various buffer conditions between pH 4.0 and 8.0 (33). Liberation of

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reducing sugar was determined using the dinitrosalicylic acid (DNS) method (32) with xylose

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as a standard. Enzyme reaction was started by mixing 50 µL of an appropriate dilution of the

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enzyme solution with 0.45 mL of substrate solution (1 %) in 50 mM sodium acetate buffer (pH

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5.0) and incubated for 10 min at 50°C. The reaction was stopped by addition of 0.75 mL of

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DNS solution and boiling for 5 min. Reducing sugars were determined by measuring the

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absorption at 540 nm. One unit of xylanase activity was defined as the quantity of enzyme

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required to liberate 1 µmol of xylose equivalent per min. For the estimation of kinetic

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parameters, the initial reaction rates of the enzymes were measured at substrate concentrations 7 ACS Paragon Plus Environment

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ranging from 0.1 to 5 mg/mL. The kinetic parameters Km and kcat of the enzymes

were

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calculated by the nonlinear least-squares method (34). Optimum temperature for the xylanase

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activities of the mutants were examined at temperatures ranging between 45 and 75°C. The

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activity was assessed by incubating the enzyme (1.0 mg/mL) in 100 µL of 50 mM sodium

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acetate buffer (pH 5.0) containing 50 mM NaCl for 10 min at the respective temperatures.

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Differential scanning calorimetry (DSC).

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Thermal stability of the enzymes was examined using differential scanning

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calorimetry (DSC). The mutants in 50 mM sodium acetate buffer (pH 5.0) were used at a final

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concentration of 1.0 mg/mL. A nanoDSC instrument (TA Instruments) was used at a scanning

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speed of 60°C/h. Control runs in the absence of protein were carried out before and after each

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sample run. DSC scans in the presence of protein were performed two or three times for each

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protein examined. DSC scans for the proteins was also carried out with xylobiose (competitive

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inhibitor of xylanase; X2) (36 mM).

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Crystallization.

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The purified thermostable mutant was dialyzed against 20 mM Tris-HCl (pH 8.0)/50

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mM NaCl and then concentrated to 13 mg/mL using Vivaspin 20-10K centrifugal filter units

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(GE Healthcare). Initial crystallization screening of the mutant protein was performed

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manually using the crystallization screening kits of Crystal Screen HT (Hampton Research),

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JCSG-plus (Molecular Dimensions) and Wizard Classic 1 and 2, 3 and 4 (Rigaku) 8 ACS Paragon Plus Environment

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crystallization regents according to the sitting-drop vapor diffusion method at 293 K in 96-well

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plates. Each drop (0.5 µL) was mixed with 0.5 µL of reservoir solution and then equilibrated

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with 60 µL of reservoir solution. After a week, well-formed crystals were obtained using

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JCSG-plus condition no. 20 (10 % [w/v] polyethylene glycol 8,000, 0.1 M Tris-HCl [pH 7.0],

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0.2 M magnesium chloride), and was further optimized to 13 % (w/v) polyethylene glycol

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8,000, 0.1 M sodium citrate (pH 5.0), and 0.2 M magnesium chloride. Optimized drops were

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obtained by mixing 1 µL of the protein solution with 1 µL of reservoir solution (2 µL total) and

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equilibrating with 1 mL of reservoir solution using the hanging-drop vapor diffusion method at

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293 K in 24-well plates.

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X-ray data collection.

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Crystals obtained as described above were soaked in cryoprotectant solution

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consisting of 30 % (v/v) ethylene glycol, 13 % (w/v) polyethylene glycol 8,000, 0.1 M sodium

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citrate (pH 5.0), and 0.2 M magnesium chloride. The soaked crystals were then collected with a

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cryo-loop and flash-cooled under a cryo-stream of nitrogen gas at 100 K. X-ray data were

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collected at the SPring-8 BL44XU (Hyogo, Japan). The dataset was collected at a single

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wavelength of 0.9 Å using a Rayonix MX300HE detector. The crystal was rotated 200° with an

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oscillation angle of 0.5° per frame (total of 400 frames). The distance from the crystal to the

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detector was 300 mm. Data were processed using the HKL2000 software package (35).

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Structure solution and refinement. 9 ACS Paragon Plus Environment

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Crystals of XylCmt9 were prepared according to the hanging-drop vapor diffusion

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method, as described above. The initial phase of the XylCmt9 structure was determined by

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molecular replacement with PHASER (36) using WT structure (PDB # 3WP3) as the search

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model. Manual adjustment of the models and model refinement were carried out using COOT

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(37), and CNS (38) and REFMAC5 (39). Water molecules were added to the models using

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ARP/wARP (40), and the 2Fo-Fc and Fo-Fc maps were then manually inspected. The

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stereochemical properties of the models were assessed using PROCHECK (41) and the

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validation tools of COOT. Figures were created using CCP4MG (42).

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RESULTS

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Structure-based sequence homology of various GH11 xylanases.

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Seven ORFs exhibiting high homology to GH11 xylanase have been identified in

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the T. cellulolyticus genome (43). In the seven ORFs, six GH11 xylanases were recently

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cloned and characterized by Watanabe et al. (22). They reported that the six xylanases

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showed similar optimum temperature and pH for enzyme activity. In the six xylanases,

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TcXylC exhibits the highest activity and its crystal structure was determined (23). Therefore,

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TcXylC was selected as the construction of thermophilic xylanase in this study. TcXylC

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consists of 14 ß-sheets (designated A2 through A6 and B1 through B9) and one α-helix, as

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shown in Figure 1. TcXylC exhibits 30~60 % amino acid sequence identity with other

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xylanases. Alignment analysis indicates that most of the sequences are similar and include

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two catalytic glutamic acid residues (Fig. 1). Watanabe et al. (22) suggested that the 10 ACS Paragon Plus Environment

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N-terminus (Met1-Arg33) of TcXylC (Fig. 1) serves as a signal sequence of the enzyme.

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Furthermore, the side chain of Gln34 faces completely outward in the structure of TcXylC.

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Therefore, we decided to remove the N-terminus of TcXylC up to Glu34 for the preparation

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of the recombinant enzymes. The recombinant TcXylC without the N-terminus up to Gln34

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was prepared and produced by the previous methods described in the Experimental

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Procedures. The activity of enzyme was not influenced by the above modification and the

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enzyme was designated as WT_TcXylC. It was reported that some of the alkalophilic

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xylanases were activated by metal ions (44, 45). In the case of TcXylC and their mutants,

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however, the activity was not affected by the presence of EDTA (10 mM) and Mg2+ (2.0 - 50

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mM) (data not shown).

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Structure-based targeting of site-directed mutagenesis sites for constructing a

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thermophilic xylanase.

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Analysis of the TcXylC crystal structures determined (23) revealed that the two

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different conformations (open and closed forms) were observed in the asymmetric unit. In the

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closed form, the N-terminal region exhibits high B-factor values (23). The region with high

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B-factor values is the flexible and/or unstable region in the protein. For constructing a

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thermostable mutant, it was key to improve the stability of the above structures. The

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substrate-binding region is important for enzyme activity. Therefore, we focused on the

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N-terminal region for constructing thermostable mutants using the structure of the closed


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form of TcXylC (PDB; 3WP3) (23). The target mutation sites for the purpose were indicated

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in Fig. 2.

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In the N-terminus of WT, Ser35 is located close to Thr62 (neighboring ß-strand)

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(Fig. 2). The distance between Ser35 and Thr62 is ideal for formation of a disulfide bond via

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S35C/T62C substitutions (their Cα-Cα and Cß-Cß distances are estimated to be 4.43 Å and

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4.77 Å, respectively (23)). Therefore, we attempted to introduce a disulfide bond via these

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substitutions. In the case of SspXyl, hydrophobic interactions between Met67 and Leu69 at

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the A2 ß-sheet are observed (46). These residues are not conserved at the N-terminus of the

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ß-sheet (A2) (Fig. 1) in TcXylC. Met67 and Leu69 in SspXyl correspond to Tyr61 and Asn63

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in TcXylC, respectively (Fig. 1). Therefore, we constructed the mutants in which Tyr61 and

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Asn63 were replaced with Met and Leu residues (Y61M and N63L), respectively in order to

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increase the C-C contact in the corresponding region in TcXylC. At the position of Asn44, at

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least one or two hydrogen bond(s) with the backbone of the adjacent strand (B2) would be

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expected to form by substitution of Asn44 with a His residue (N44H) (Fig. 2A). Proline

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residue is known to impart a rigid and stable conformation to ß-turn or loop structures (47-49).

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Regarding selection of a site for introducing a Pro residue to enhance the stability, we

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selected Asp65, which is located in the loop region (between A2 and A3) (Fig. 2A). Sequence

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homology analysis (Fig. 1) indicated that Asp65-Asn66 of TcXylC corresponds to

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Pro31-Gly32 of TrXyl. We therefore performed D65P and N66G substitutions at the

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N-terminus of the ß-strand (A2) of TcXylC. From the sequence homology, furthermore, Pro

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is observed at the position corresponding to Thr101 of TcXylC (located in the loop region 12 ACS Paragon Plus Environment

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between A5 and B5) in some xylanases (Fig. 1). Therefore, we introduced a Pro residue at the

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position of Thr101 in the loop region (Fig. 2A). Based on sequence homology, we introduced

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T101P and S102N substitutions in TcXylC (A5) (Fig. 2A). A typical ß-turn was observed at

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Asp65-Asn66 but not at Thr101-Ser102.

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The specific activity and temperature dependence of activity were examined for

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each mutant. For all mutants prepared, the significant change of the specific activities at 45°C

260

were not observed (Table 1). The temperature dependencies of the enzyme activity were

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examined at different temperatures. Fig. 3 shows the temperature dependent activity of the

262

mutants. For Y61M, N63L, D65P and N66G mutants, significant instability was observed at

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60°C in comparison with WT (Fig. 3). However, The Y61M/N63L and D65P/N66G double

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mutations retain their activities at 60°C (Fig. 3). For the other mutants (S35C, N44H, T62C,

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T101P, and S102N and S35C/T62C), a decrease in the activity at 60°C was not observed (Fig.

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3). Therefore, the temperature dependent activity (45-75°C) for the mutants excluding Y61M,

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N63L, D65P and N66G was examined. The results indicate that Y61M/N63L, D65P/M66G,

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S35C, N44H, T62C, S35C/T62C, T101P, and S102N mutations shifted the temperature

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optima of the enzymes (Fig. 3). Thermal stability and the melting temperatures (Tm) of WT

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and mutant enzymes were also examined using DSC (Table 1, Fig. 4). Table 1 shows the

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values for melting temperatures (Tm), ∆H, and ∆S for heat denaturation process. The result

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that the Tm values of the WT, S102N, and S35C/T62C TcXylC mutants were estimated to be

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58.9, 60.0, and 74.5°C, respectively indicating that S102N, D65P/M66G, and S35C/T62C

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TcXylC mutants enhanced the thermostability of the enzyme (Table 1, Fig. 2B). However, for 13 ACS Paragon Plus Environment

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the other mutants, improvements in the thermostability corresponding to Tm of the enzyme

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were not observed (Table 1, Fig. 4). Thermal stability of WT and mutant enzymes with the

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substrate analogue xylobiose (X2) (36 mM) was also examined using DSC (the detailed data

278

not shown). Most of the values of Tm were increased (Table 1).

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For the S35C/T62C substitution, the relative activity at the high temperature was

280

markedly increased (Fig. 3). Furthermore, the Tm of the S35C/T62C mutant was over 15°C

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higher than that of the WT enzyme (Table 1, Fig. 4). However, the S35C and T62C single

282

substitution mutants did not at over 65°C compared with WT (Fig. 3). The above result

283

suggests that one disulfide bond is introduced in the enzyme via the S35C/T62C substitution

284

and contributes to the thermostability greatly.

285

Thus, we decided to introduce a total of nine point mutations that enhanced the

286

activity of the enzyme. The mutant gene, designated XylCmt9 (encoding S35C, N44H, Y61M,

287

T62C, N63L, D65P, N66G, T101P, and S102N substitutions) was constructed as described

288

above in the Experimental Procedures. The prepared XylCmt9 mutant produced the highest Tm

289

(80.5°C) (Table 1, Fig. 4) of all of the mutants examined, without any loss in original specific

290

activity (Fig. 3).

291 292

Structure of thermostable xylanase (XylCmt9).

293

In order to examine the structural basis of the enhanced thermostability of the

294

enzyme, we analyzed the crystal structure of the XylCmt9 variant. Crystals of XylCmt9 were

295

prepared (approximately 300 × 300 × 10 µm in size) and the structure was determined, as 14 ACS Paragon Plus Environment

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Biochemistry

296

described in the Experimental Procedures. The prepared crystals belonged to space group P21,

297

with unit cell a = 75.9 Å, b = 179.8 Å, c = 93.3 Å, and ß = 103.8°. Data collection and

298

refinement statistics are shown in Table 2. In the asymmetric unit, 12 molecules of XylCmt9

299

were identified; the Matthews coefficient (50) (VM) was calculated as 2.50 Å3 Da−1, with a

300

solvent content of 50.9 % (v/v). After refinement, R factors of the model were estimated as

301

Rwork = 18.0 % and Rfree = 22.8 %. A Ramachandran plot (51) for the model showed that 94.8 %

302

of the residues were in the most favored regions, with 4.4 % of the residues in additional

303

allowed regions. The coordinates and structure factors for XylCmt9 were deposited in Protein

304

Data Bank under the accession code 5HXV. The overall structures of 12 molecules of XylCmt9

305

were built from Cys35 to Ser223, and the root mean square deviation (RMSD) was less than

306

0.47 Å over 189 Cα atoms. The RMSD value indicated that the overall structures of the 12

307

molecules of XylCmt9 were almost identical (Fig. 5A). It was determined that the overall

308

structure of XylCmt9 was almost identical to that of the WT except for the N-terminal region.

309

In spite of high amounts of magnesium chloride (200 mM) in the crystallization reagent, the

310

significant electron density map corresponding to Mg2+ was not observed. All nine mutations

311

were identified in the structure. The chain A molecule of the 12 molecules (PDB; 5HXV)

312

exhibited relatively low B-factor values. Superimposition of chain A and closed form of WT

313

showed the identity of the whole structure (Fig. 5B). It was difficult to distinguish the

314

difference between open and closed forms in XylCmt9. Using the chain A molecule, therefore,

315

the difference of the N-terminal region was examined. As expected from the above result, the

316

disulfide bond between Cys35 and Cys62 was observed in XylCmt9 (Fig. 6A). Their Cα-Cα 15 ACS Paragon Plus Environment

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317

and Cß-Cß distances were changed and the N-terminal region was slightly pulled into the

318

molecule by the newly introduced disulfide bond. It is difficult to compare the B-factor values

319

between the different crystals (WT and XylCmt9). However, the B-factor values of the region

320

were relatively low in XylCmt9. In addition, the C-C contacts between Ile36 Cδ1 and Leu63 Cδ1

321

(bond length = 3.77 Å) and between Met61 Cε and Leu63 Cδ2 (bond length = 3.65 Å) in A2 were

322

observed as good bond in XylCmt9 (Fig. 6A). Both Met61 and Leu63 were found to be essential

323

for the formation of a hydrophobic network around the N-terminus when the disulfide bond was

324

formed (Fig. 6A). As expected, the imidazole ring of His44 Nδ1 atom and the O atom of the

325

Gly64 main chain (neighboring strand A2) is found to form a new hydrogen bond at 3.23 Å in

326

XylCmt9 as a result of the N44H substitution (Fig. 6B). At a ß-turn (between A2 and A3),

327

Pro65 and Gly66 were observed without the conformational change in the region. At the loop

328

region (between A5 and B5), Pro101 and Asn102 were observed with the formation of new

329

hydrogen bonds between the N and O atoms of Asn102 and Ser215 main chain in the

330

neighboring ß-strand (A4) (Fig. 6C).

331 332

Characterization of the mutants.

333

Xylanase activity was measured by incubating the enzyme at 50°C using 1 % (w/v)

334

birch-wood xylan as the substrate. XylCmt9 showed an optimum pH of 4.0 - 5.0, similar to

335

WT. Compared with WT, a slight increase in the specific activity was observed for XylCmt9

336

(Table 3). The kinetic parameters Km and kcat of the enzymes were calculated by the nonlinear


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Biochemistry

337

least-squares method (Table 3). The increased value of kcat may be due to a faster rate of

338

product release, and the higher mobility may be associated with higher activity.

339 340

DISCUSSION

341

In this study, we constructed thermostable mutants of TcXylC using protein

342

engineering methods and examined the structural basis for thermostabilization. Crystal

343

structure analysis revealed two conformers of TcXylC structure (open and closed forms) (23).

344

The RMSD between closed and open forms were estimated to be 0.55 Å over 189 Cα (23).

345

The structure of the closed form may mimic the forming enzyme–substrate complex. In the

346

closed form, the N-terminal region, located far from the catalytic site, exhibits high B-factor

347

values and appeared to be a suitable target for the construction of a thermostable xylanase.

348

From BRENDA database (52), one of the xylanases (EC 3.2.1.8) has observed the activation

349

in the presence of Mg2+ (maximum 6 mM) (45). In the case of TcXylC and their mutants,

350

however, the activity was not affected by the presence of EDTA and Mg2+ (data not shown).

351

Furthermore, the significant electron density map corresponding to Mg2+ was not observed in

352

its crystal structure. From these results, it was clarified that TcXylC was not activated by the

353

bound metal ions.

354

Thus, we focused on stabilizing the N-terminal region to enhance the

355

thermostability. The effects of the mutations on the thermostability were evaluated based on

356

the Tm values determined by DSC, and the temperature dependence of the activity for each

357

mutant. Result of Tm values determined by DSC and the enzyme activity measurement 17 ACS Paragon Plus Environment

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Page 18 of 47

358

experiments differed. Most of the values of Tm of the mutants were lower than 60°C (WT)

359

(Table 1, Fig. 4). For the mutants excluding Y61M, N63L, D65P, and N66G, however,

360

significant increases in activity were observed at 65°C (Fig. 3). The result suggests that the

361

enzyme is stabilized by the substrate. Therefore, thermal stability of the enzymes with the

362

substrate analogue xylobiose (X2) was measured by DSC. Most of the values of Tm of the

363

mutants were increased by adding X2 (36 mM) (Table 1). The increased values of Tm of the

364

mutants are dependent on the ligand binding (53). However, disagreement between the values

365

of Tm by DSC and activity measurement experiments was also observed. Similar

366

disagreement was observed in the case of xylanase mutants from B. circulans (30). Therefore,

367

it is speculated that the protein unfolding process estimated by DSC is not necessarily same as

368

the thermostabilization of the enzyme.

369

We gave priority to the kinetic results obtained from the enzyme activity temperature

370

dependence experiments. In the mesophilic xylanase SspXyl, a significant C-C contact is

371

observed between Met67 and Leu69 in the structure of N-terminal region (46). We have no

372

structural information of Y61M/N63L. From the analysis of the crystal structure of XylCmt9

373

(Fig. 6A), however, the expected C-C contact between Met61 Cε and Leu63 Cδ2 seems to

374

contribute the thermostability in Y61M/N63L. Five ß-turns (Asn44-Asn45-Gly46-Tyr47,

375

between

376

Ser132-Ser133-Gly134-Leu135, between B6 and B9; Ser143-Asn144-Gly145-Gly146,

377

between B9 and B8; and Ser160-Ile161-Glu162-Gly163, between B8 and B9) were observed in

378

the structure of WT (Fig. 1). The region Asp65-Asn66-Gly67-Glu68 forms a typical ß-turn I.

B1

and

B2;

Asp65-Asn66-Gly67-Glu68,


between

A2

ans

A3;

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Biochemistry

379

The single substitution mutations D65P and N66G reduced the Tm. But in the double mutant

380

(D65P/N66G), the Tm increased relative to WT (Table 1). The activity of the double mutant

381

(D65P/N66G) was also higher than that of WT at 65°C (Fig. 3). In the region, Asn66 was

382

observed in an allowed but not preferred region in the Ramachandran plot. Therefore, it was

383

assumed that a stable conformation in the turn structure was formed due to the combination of

384

the introductions (Pro65 and Gly66), thus enhancing the enzyme’s thermostability. In addition,

385

the result that the value of ∆H by DSC was increased by the mutation (Table 1) suggests that the

386

improved thermostability is also derived by the C-C contact of the introduced Pro. From the

387

theoretical analysis of the ß-turn I structure, however, the Pro introduction seemed to be

388

preferable at the position of Asn66 for its stability (47). The detailed analysis and experiment

389

for this position are in progress. The loop region containing Thr101 and Ser102 does not form a

390

typical ß-turn. From the crystal structural analysis for XylCmt9, it is elucidated that the

391

substitution of Thr101 with a Pro residue contributes to the C-C contact to the side chain of

392

Phe99 and the formation of the main chain in the neighboring ß-strand (B4) (Fig. 6C).

393

Therefore, T101P or S102, or both mutations may have induced the conformational change in

394

the turn region (between A5 and B5), located in an area accessible to the surface, making it

395

possible to couple two hydrogen bonds. These structural features are in agreement with the

396

results of DSC experiments and temperature dependence experiments, in which thermostability

397

increased slightly (Table 1). Interestingly, the five ß-turns observed in WT contain no Pro

398

residues. Therefore, we have a chance to improve its thermostability more by introducing Pro at

399

the sites. 19 ACS Paragon Plus Environment

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400

By introducing a disulfide bond between Ser35 and Thr62 in TcXylC (via

401

S35C/T62C substitutions), we constructed a mutant with greater thermostability (Fig. 3, 4). The

402

effect of introducing a disulfide bond in the flexible N-terminal region of AoXyn11A was

403

discussed in a recent report (27). Based on molecular dynamics simulation, it has been

404

estimated that one disulfide bond stabilizes the structure and increases the thermostability of

405

5°C. In the present study, however, we were able to increase the optimum temperature by more

406

than 10°C relative to the WT enzyme by introducing one disulfide bond. We have no

407

information for the structure of the S35C/T62C mutant. However, the positions of the two

408

cysteines (Cys35-Cys62) selected were more suitable sites for stabilizing the enzyme. The

409

value of ∆H by DSC was not improved by the disulfide bond (Table 1). Therefore, the effect of

410

the thermostability seems to be controlled by the entropic effect of the disulfide bond

411

introduced.

412

The crystal structural analysis for XylCmt9 showed not only the formation of a

413

disulfide bond between Cys35 and Cys62, but also that a newly induced C-C contact contribute

414

to the thermostability of the enzyme (Fig. 6A). The main chain Cα of Ile36 moved significantly

415

towards the interior of the protein (1.31 Å) and the side chain flipped toward Leu63 resulting

416

conformational changes allowed a new C-C contact to occur between the Cδ1 of Ile36 and the

417

Cδ1 of Leu63 (Fig. 6A). Furthermore, it was also possible that a new contact formed between

418

Met61 Cε and Leu63 Cδ2 (Fig. 6A). Thus, it was elucidated that the formation of a new

419

hydrophobic contact was triggered by the introduction of the disulfide bond. In the N44H

420

mutant, a new hydrogen bond between the side chain of His44 and the Nδ1 and O atoms of the 20 ACS Paragon Plus Environment

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Biochemistry

421

Gly64 main chain was observed (Fig. 6B). Table 1 shows that the values of ∆H by DSC for

422

Y61M/N63L and D65P/N66G mutants were increased (+22 and +63 kJ/mol) compared with

423

WT. On the other hand, the values of ∆H by DSC for T101P, S102N and N44H mutants were

424

decreased (-45, -37 and -85 kJ/mol). Therefore, the increased value (+87 kJ/mol) for ∆H and

425

value (+0.5 kJ/K mol) for ∆S for XylCmt9 also supports that the formation of new hydrogen

426

bonding and C-C contacts enhanced by the introduced disulfide bond contribute to

427

thermostability. The positive effects of the mutations seem to be partially additive (54). The

428

study mentioned when the increases in activity by a double mutant are greater than the

429

individual increases observed for each single mutation attributes to a cooperative interaction

430

between the two residues mutated (54). Similar phenomena were observed in our study.

431

In this study, we not only enhanced the thermostability of TcXylC to 80.5°C by

432

site-directed mutagenesis, also elucidated the structural basis of thermostability. Currently, we

433

are evaluating the application of thermostable XylCmt9 to the degradation of actual biomass

434

sources, such as bagasse and rice husks.

435 436

AUTHOR INFORMATION

437

Corresponding Author

438

Tel.: +81-82-420-8258, Fax: +81-82-420-7820, E-mail: [email protected]

439 440

Funding

441

This work was supported by the Japan Science and Technology Agency (JST)-Advanced Low

442

Carbon Technology Research and Development Program (ALCA) (DGA20264004). 21 ACS Paragon Plus Environment

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443 444

ACKNOWLEDGEMENTS

445

X-ray diffraction data were obtained using the beam line BL44XU in SPring-8, Hyogo, Japan,

446

with approval of the Institute for Protein Research, Osaka University, Osaka, Japan (proposal

447

No. 2014A6903 and 2014B6903). This work was supported by Toray Industries Inc. New

448

Frontiers Research Laboratories, (Kanagawa, Japan). We would like to thank Dr. Han-woo

449

Kim and Ms. Yuka Maeno a former member at National Institute of Advanced Industrial

450

Science and Technology (AIST) for the preliminary experiment of constructing thermostable

451

xylanases. K. Ishikawa thanks Dr. Wing Sung of National Research Council Canada and Dr.

452

Jeffrey Tolan of Iogen Co. (Ottawa, ON Canada) for giving him a chance to participate in the

453

pulp breaching project using xylanase supported by G7 summit fellowship program in Canada.

454 455

ABBREVIATIONS USED

456

GH, glycoside hydrolase; TcXylC, xylanase 11C from T. cellulolyticus; DNS, dinitrosalicylic

457

acid; DSC, differential scanning calorimetry; RMSD, root mean square deviation.

458 459

REFERENCES

460

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461 462

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Benamrouche, S., Debeire, P., O'Donohue, M., and Chabbert, B. (2004) Impact and

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structure of a GH10 xylanase from Fusarium oxysporum reveals the presence of an


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extended loop on top of the catalytic cleft. Acta Crystallogr., Sect. D: Biol. Crystallogr.

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14. Harris, G. W., Jenkins, J. A., Connerton, I., Cummings, N., Loleggio, L., Scott, M.,

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Hazlewood, G. P., Laurie, J. I., Gilbert, H. J., and Pickersgill, R. W. (1994) Structure of the

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Langan, P., Graham, D., and Kovalevsky, A. (2014) X-ray crystallographic studies of

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mechanism. Acta Crystallogr., Sect. D: Biol. Crystallogr. 70, 11-23.

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19. Fujii, T., Hoshino, T., Inoue, H., and Yano, S. (2014) Taxonomic revision of the

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protein using bicinchoninic acid. Anal. Biochem. 150, 76-85.

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32. Bailey, M. J., Biely, P., and Poutanen, K. (1992) Interlaboratory testing of methods for assay of xylanase activity. J. Biotechnol. 23, 257-270. 33. McIlvaine, T. C. (1921) A buffer solution for colorimetric comparison. J. Biol. Chem. 49, 183-186.

558

34. Sakoda, M., and Hiromi, K. (1976) Determination of the best-fit values of kinetic

559

parameters of the Michaelis-Menten equation by the method of least squares with the

560

Taylor expansion. J. biochem. 80, 547-555.

561 562

35. Otwinowski, Z., and Minor, W. (1997) Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307-326.

563

36. McCoy, A. J., Grosse-Kunstieve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C., and

564

Read, R. J. (2007) Phaser crystallographic software. J. Appl. Crystallogr. 40, 658-674

565

37. Emsley, P., and Cowtan, K. (2004) Coot: model-building tools for molecular graphics.

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Acta Crystallogr., Sect. D: Biol. Crystallogr. 60, 2126-2132. 26 ACS Paragon Plus Environment

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38. Brünger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R.

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W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M.,

569

Simonson, T., and Warren, G. L. (1998) Crystallography & NMR System: a new software

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suite for macromolecular structure determination. Acta Crystallogr., Sect. D: Biol.

571

Crystallogr. 54, 905-921.

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39. Murshudov, G. N., Skubák, P., Lebedev, A. A., Pannu, N. S., Steiner, R. A., Nicholls, R.

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A., Winn, M. D., Long, F., and Vagin, A. A. (2011) REFMAC5 for the refinement of

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macromolecular crystal structures. Acta Crystallogr., Sect. D: Biol. Crystallogr. 67,

575

355-367.

576

40. Gerrit, G. L., Serge, X. C., Victor, S. L., and Anastassis, P. (2008) Automated

577

macromolecular model building for X-ray crystallography using ARP/wARP version 7.

578

Nat Protoc. 3, 1171-1179.

579

41. Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993)

580

PROCHECK: a program to check the stereochemical quality of protein strucutres. J. Appl.

581

Crystallogr. 26, 283-291.

582

42. Potterton, L., McNicholas, S, Krissinel, E., Gruber, J., Cowtan, K., Emsley, P., Murshudov,

583

G. N., Cohen, S., Perrakis, A., and Noble, M. (2004) Developments in the CCP4 molecular

584

graphics project. Acta Crystallogr., D: Biol. Crystallogr. 60, 2288-2294.

585

43. Fujii, T., Koike, H., Sawayama, S., Yano, S., and Inoue, H. (2015) Draft Genome

586

Sequence of Talaromyces cellulolyticus Strain Y-94, a Source of Lignocellulosic

587

Biomass-Degrading

588

10.1128/genomeA.00014-15.

Enzymes.

Genome

Announc.

3,

pii:

e00014-15.

doi:

589

44. Ratanakhanokchai, K., Kyu, K. L., and Tanticharoen, M. (1999) Purification and

590

properties of a xylan-binding endoxylanase from Alkaliphilic bacillus sp. strain K-1. Appl.

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Environ. Microbiol. 65, 694-697. 27 ACS Paragon Plus Environment

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45. Manikandan, K., Bhardwaj, A., Gupta, N., Lokanath, N. K., Ghosh, A., Reddy, V. S., and

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Ramakumar, S. (2006) Crystal structures of native and xylosaccharide-bound alkali

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thermostable xylanase from an alkalophilic Bacillus sp. NG-27: structural insights into

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alkalophilicity and implications for adaptation to polyextreme conditions. Protein Sci. 8,

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1951-1960.

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46. Wouters, J., Georis, J., Engher, D., Vandenhaute, J., Dusart, J., Frere, J. M., Depiereux, E.,

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and Charlier, P. (2001) Crystallographic analysis of family 11 endo-beta-1,4-xylanase

599

Xyl1 from Streptomyces sp. S38. Acta Crystallogr., Sect. D: Biol. Crystallogr. 57,

600

1813-1819.

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47. Matthews, B. W., Nicholson, H., and Becktel, W. J. (1987) Enhanced protein

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thermostability from site-directed mutations that decrease the entropy of unfolding. Proc.

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Natl. Acad. Sci. U. S. A. 84, 6663-6667.

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48. Trevino, S. R., Schaefer, S., Scholtz, J. M., and Pace, C. N. (2007) Increasing protein

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conformational stability by optimizing beta-turn sequence. J. Mol. Biol. 373, 211-218.

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49. Chou, P. Y., and Fasman, G. D. (1974) Conformational parameters for amino acids in

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helical, beta-sheet, and random coil regions calculated from proteins. Biochemistry 13,

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211-222.

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50. Matthews, B. W. (1968) Solvent content of protein crystals. J. Mol. Biol. 33, 491-497.

610

51. Ramachandran, G. N., and Sasiskharan, V. (1968) Conformation of polypeptides and

611 612 613

proteins. Adv. Protein Chem. 23, 283-438. 52. Schomburg, I., Chang, A., and Schomburg, D. (2002) BRENDA, enzyme data and metabolic information. Nucl. Acids Res. 30, 47-49.

614

53. Schellman, J. A. (1975) Macromolecular binding. Biopolymers 14, 999-1018.

615

54. Mildvan, A. S., Weber, D. J., and Kuliopulos, A. (1992) Quantitative interpretations of

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double mutations of enzymes. Arch. Biochem. Biophys. 294, 327-340. 28 ACS Paragon Plus Environment

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Biochemistry

617

55. Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) CLUSTAL W: improving the

618

sensitivity of progressive multiple sequence alignment through sequence weighting,

619

position-specific gap penalties and weight matrix choice. Nucl. Acids Res. 22, 4673-4680.

620

56. Gouet, P., Robert, X., and Courcelle, E. (2003) ESPript/ENDscript: extracting and

621

rendering sequence and 3D information from atomic structures of proteins. Nucl. Acids

622

Res. 31, 3320-3323.

623


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

624

FIGURE LEGENDS

625

Figure 1. Structure-based sequence alignments of various GH11 xylanases. Strictly conserved

626

residues are boxed in red. Similar residues are shown in black bold characters and boxed in

627

yellow. The catalytic residues are indicated with blue boxes. One α-helix and 14 ß-strands

628

(A2-6, B1-9) in TcXylC are representative as a black coil and black arrows, respectively. The

629

species producing the various xylanases are abbreviated as follows: TcXylC: GH11 from

630

Talaromyces cellulolyticus (GenBank Accession Number BAO51921), TrXyl: GH11 from

631

Trichoderma reesei (GenBank Accession Number ACB38137), ScXyl: GH11 from

632

Schizophyllum commune (GenBank Accession Number AAB29056), SoXyl: GH11 from

633

Streptomyces olivaceoviridis (GenBank Accession Number AHK22787), SspXyl: GH11 from

634

Streptomyces sp. S38 (GenBank Accession Number CAA67143), BcXyl: GH11 from Bacillus

635

circulans (GenBank Accession Number P09850), AnXyl: GH11 from Aspergillus niger

636

(GenBank Accession Number AAS46913), and NpXyl: GH11 from Neocallimastix

637

patriciarum (GenBank Accession Number 3WP4_A). The figure was constructed using

638

ClustalW (55) and ESPript (56).

639 640

Figure 2. Mutations mapped on the closed form of WT_TcXylC. A is colored with increased

641

the Tm by 0-5 degrees (yellow; S102), by 5-10 degrees (cyan; D65/N66), by 10-15 degrees (red;

642

S35/T62). B is colored with increased activity by single mutation sites (yellow; S35, N44, T62,

643

T101 and S102), by double mutation sites (cyan; Y61/N63 and D65/N66). Each residue was

644

drawn on the WT_TcXylC structure (colored white).

645 646

Figure 3. Relative activities of WT and mutants. Optimum temperature of all proteins (1.0

647

mg/mL) was measured at temperatures ranging from 45 to 60-75°C using the DNS method in

648

50 mM sodium acetate (pH 5.0). Samples were pre-heated for 10 min at the indicated 30 ACS Paragon Plus Environment

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Biochemistry

649

temperature before mixing with the substrate. Each sample is shown as follows (WT: white

650

squares with dashed line; S35C: open diamonds; N44H: white squares; Y61M: black bars;

651

T62C: open triangles; N63L: black crosses; D65P: black squares with dashed line; N66G: black

652

triangles with dashed line; T101P: black crosses; S102N: black squares; S35C/T62C: black

653

triangles; Y61M/N63L: black diamonds; D65P/N66G: black circles; XylCmt9: open circles).

654

In both figures, the percent relative activity is based on the specific activity (U/mg) of XylCmt9

655

at 75°C. The data plotted are averages of three experiments.

656 657

Figure 4. DSC results. The DSC curves for nine point mutations and a double mutation are

658

shown (S35C: brown; N44H: red; Y61M: green; T62C: blue; N63L: pink; D65P: purple;

659

N66G: orchid; T101P: cyan; S102N: dark green; S35C/T62C: navy; Y61M/N63L: yellow and

660

D65P/N66G: gold). WT and XylCmt9 are also shown as black and gray dotted lines,

661

respectively. All samples were adjusted to 1.0 mg/mL and dialyzed against 50 mM sodium

662

acetate buffer (pH 5.5) before the DSC measurements. Bar represents heat capacity (1 J K-1 g-1).

663 664

Figure 5. Superimposed models of 12 molecules of XylCmt9, and XylCmt9 (chain A) and WT

665

(closed form). A, 12 molecules (chain A-L, PDB: 5HXV) of XylCmt9 are indicated worm

666

models and colored by red, blue, pink, purple, yellow, light blue, cyan, green, coral, magenta,

667

gold, and tan, respectively. B, XylCmt9 (cyan) and WT (pink) are shown as ribbon models with

668

labeled ß-strands (B1-9/A2-6), one α-helix and thumb. Two glutamate residues (Glu119 and

669

Glu210) are indicated in the cylinder model. N- and C-termini are labeled.

670 671

Figure 6. New interactions in XylCmt9. A, Stereo view showing an overlay of the disulfide

672

bond and C-C contact with superimposed Ile36 of the WT enzyme. S35C, Ile36, T62C, and

673

N63L of XylCmt9 (italic) are shown as a cylinder model with atomic element colors (O atoms: 31 ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

674

red; N atoms: blue; C atoms: cyan) and the disulfide bond in yellow. WT Ile36 is also shown in

675

the cylinder model with atomic element colors (O atoms: red; N atoms: blue; C atoms: pink).

676

The side chains of Ile36 and N63L of XylCmt9 are also highlighted cyan in the cylinder model

677

and the C-C contact is indicated with a black dashed line. Ile36, Ser35, Asn62, and Asn63 of

678

WT are colored pink in the cylinder model. B, Stereo view showing an overlay of a hydrogen

679

bond. N44H and Gly64 of XylCmt9 are shown in the cylinder model with atomic element

680

colors. The hydrogen bond between N44H and Gly64 is indicated by black dashed lines. C,

681

Stereo view showing an overlay of two hydrogen bonds. T101P, S102N, and Ser215 of

682

XylCmt9 are shown in the cylinder model with atomic element colors. The two hydrogen bonds

683

between S102N and Ser215 are indicated by black dashed lines.

684 685 686

SUPPLEMENTAL Fig. 1 SDS-PAGE analysis of purified WT and XylCmt9. M: molecular

687

weight markers; lane 1: WT; lane 2: XylCmt9. Both proteins are the final product after

688

purification using a Superdex 200 16/60 gel filtration column (GE Healthcare).

689 690

SUPPLEMENTAL Table 1 Summary of nucleotide primers used in this study.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

691

Table 1 Summary of the relative activities and thermodynamic parameters for the heat

692

denaturation of TcXylC and mutants Relative activity (45°C) %

693 694 695 696 697

Tm o

Tm* (+X2)*

C

∆H(Tm)

∆S(Tm)

∆H(58.9°C)**

∆S(58.9°C)**

kJ/mol

kJ/(K mol)

kJ/mol

kJ/(K mol)

WT

100 ± 3

58.9

(59.8)

461

1.39

461

1.38

Y61M

94 ± 3

53.5

(54.1)

390

1.19

398

1.22

N63L

85 ± 3

50.6

(51.5)

349

1.08

362

1.12

Y61M/N63L

109 ± 3

56.7

(59.0)

483

1.55

486

1.56

n.d.

n.d.

n.d.

***

D65P

98 ± 3

50.0, 58.1

(58)

226

N66G

100 ± 4

49.5, 57.8

(58)

309

n.d.

n.d.

n.d.

D65P/N66G

102 ± 2

64.6

(65.4)

524

1.55

515

1.52

S35C

101 ± 2

56.6

(57.3)

413

1.25

417

1.26

T62C

86 ± 2

58.3

(57.9)

376

1.13

377

1.13

S35C/T62C

97 ± 3

74.5

(75.1)

469

1.35

445

1.28

T101P

99 ± 2

58.8

(60.5)

416

1.25

416

1.25

S102N

100 ± 2

60.0

(60.8)

414

1.24

412

1.23

N44H

101 ± 4

56.4

(57.3)

376

1.14

380

1.15

XylCmt9

114 ± 3

80.5

(81.7)

548

1.55

514

1.45

* Tm with 36 mM X2 **∆H(58.9oC) and ∆S(58.9oC) are the enthalpy and entropy changes at Tm of WT , respectively calculated by using the averaged heat capacity change ∆Cp=1.5 kJ/(K mol) and the equations of of ∂∆H/∂T= ∆Cp and ∂∆S /∂T= ∆Cp /T. *** not determined.

698 699 700 701 702 703 704 705 33 ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

706

Table 2 Data collection and refinement statistics Data collection

XylCmt9

Wavelength (Å)

0.9

Space group

P21

Unit cell: a, b, c (Å), ß (°)

a=75.9, b=179.8, c=93.3, ß=103.8

Matthews coefficient (Å3Da-1)

2.5

Solvent content (%)

50.9

Resolution (Å)

50.0-2.0 (2.03-2.00)*

Rmerge a (%)

13.0 (28.0)*

Average I/σ(I)

8.6 (2.8)*

Completeness (%)

94.2 (88.2)*

Redundancy

2.7 (2.2)*

No. molecules/asym

12

Refinement Resolution (Å)

20.0-2.0

No. reflections (test reflections)

145,667 (7,697)

Rwork b/Rfree c (%)

18.0/22.8

No. atoms

18,554

Protein

17,352

Water

1,202

Mean overall B factor (Å2)

11.2

Root mean square deviations Bond lengths (Å)

0.01

Bond angles (°)

1.88


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Ramachandran plot In most favored regions (%)

94.8

In allowed regions (%)

4.4

PDB accession #

5HXV

707

*

708

a

709

measurement of reflection hkl, including symmetry-related reflections, and is their

710

average. b Rwork = ΣhΣi ||Fo| – |Fc|| / Σ |Fo|. c Rfree is Rwork for approximately 5 % of the reflections

711

that were excluded from the refinement.

Outer shell (2.03-2.00 Å) Rmerge = ΣhklΣi | Ii(hkl) – | Σ hklΣiIi(hkl), where Ii(hkl) is the intensity of the ith

712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 35 ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

728

Page 36 of 47

Table 3 Kinetic parameters of WT and XylCmt9 Enzyme (50°C)

Specific activity (U/mg)

Km (%)

kcat (sec−1)

kcat / Km (%-1 sec-1)

WT

3,991

4.7 ± 0.1

35.3 ± 0.3

7.5

XylCmt9

4,659

7.1 ± 0.1

49.8 ± 0.2

7.0

729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 36 ACS Paragon Plus Environment

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

750

For Table of Contents Use Only

751

Manuscript Title: Construction of thermophilic xylanase and its structural analysis

752

Authors: Masahiro Watanabe, Harumi Fukada and Kazuhiko Ishikawa

753

754 755 756 757 758 759 760 761 762 763 764 765 766 767 768


Biochemistry

Page 38 of 47 B1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

A2

A3

B5

B3

A5

B6

B7

B9

A6

B2

α-helix

B8

B4

A4

ACS Paragon Plus Environment

Fig. 1 Watanabe et al.

Page 39 of 47

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Biochemistry

D65

T62

S35

S102

N66 N44

N63

Y61 T101


Fig. 2A Watanabe et al.

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

D65

T62

Page 40 of 47

S35

S102

N66


Fig. 2B Watanabe et al.

Page 41 of 47

WT

100

S35C N44H 80

Y61M T62C

Relative activity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Biochemistry

N63L

60

D65P N66G 40

T101P S102N S35C/T62C

20

Y61M/N63L D65P/N66G 0 45

50

55

60

65

70

75

XylCmt9

Temperature (℃)



Biochemistry

1 J/(K g )

XylCmt9 S35C/T62C S102N WT

Heat Capacity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 42 of 47

T101P T62C S35C N44H Y61M Y61M/N63L N63L N66G D65P

30

40

50

60

70

D65P/N66G

80

90

o

Temperature / C



Page 43 of 47

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Biochemistry



Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

N

Page 44 of 47

A2 A3

B1 B2

C B3

Thumb

A5

A4

A6

B4 Glu210 B5 Glu119

B6 B7 B8

α-helix


B9


Page 45 of 47

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Biochemistry

Tyr61

Y61M

Tyr61

Y61M

3.6

3.6 Asn63

Ile36

Asn63 Ile36

3.7 N63L

Ile36

T62C

3.7 N63L

Ile36

T62C

Thr62

S35C (N-ter.)

Thr62

S35C (N-ter.)

Ser35

Ser35



Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 46 of 47

Gly64

3.2

Gly64

3.2

Gly64

Asn44

Gly64

Asn44

N44H

N44H



Page 47 of 47

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Biochemistry

Ser102

S102N

Ser215

3.3

Ser102

S102N

Ser215

Thr101

3.3

Thr101 2.8

2.8 Ser215

Ser215 T101P


T101P

Fig. 6C Watanabe et al.

Construction of Thermophilic Xylanase and Its Structural Analysis

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