Crystallization and preliminary X-ray crystallographic analysis of a novel α-l-arabinofuranosidase (CtGH43) from Clostridium thermocellum ATCC 27405

A truncated family 43 glycoside hydrolase module (CtGH43) from C. thermocellum ATCC 27405 was purified and crystallized. Data were collected to 1.1 and 1.65 Å resolution in monoclinic and cubic space groups, respectively.

Abstract

The truncated carbohydrate-active enzyme belonging to family 43 glycoside hydrolase from Clostridium thermocellum (CtGH43) is an α-l-arabinofuranosidase that in combination with endoxylanase leads to complete breakdown of l-arabinosyl-substituted xylans. The recombinant enzyme CtGH43 from C. thermocellum was overexpressed in Escherichia coli and purified by immobilized metal-ion affinity chromatography. The recombinant CtGH43 has a molecular mass of 35.86 kDa. Preliminary structural characterization was carried out on CtGH43 crystallized from different conditions, which gave either cube-shaped or brick-shaped crystals. These diffracted to a resolution of 1.65 Å for the cubic form and 1.1 Å for the monoclinic form. Molecular replacement was used to solve the CtGH43 structure.

1. Introduction  

α-l-Arabinofuranosidase (α-l-Araf; EC 3.2.1.55) catalyzes the hydrolysis of l-arabinosyl linkages attached to the β-1,4-xylo­pyranose backbone (Saha, 2003 ▶). The latest classification based on amino-acid sequences, primary-structure similarities and hydrophobic cluster analysis classifies α-l-arabinofuranosidases (α-l-Araf) into five glycoside hydrolase (GH) families, i.e. GH30, GH43, GH51, GH54 and GH62 (Zhou et al., 2012 ▶; Cartmell et al., 2011 ▶; Sørensen et al., 2006 ▶; Guais et al., 2010 ▶; Hashimoto et al., 2011 ▶). Although all GH43 family members belong to clan GH-F and have a fivefold β-propeller core architecture, they have different activities (see the CAZy website, http://www.cazy.org; Lombard et al., 2013 ▶).

The truncated 903 bp gene encoding the N-terminus of CtGH43 (family 43 glycoside hydrolase from Clostridium thermocellum) is part of a full-length family 43 glycoside hydrolase (Ct43Araf) which also contains two tandem family 6 carbohydrate-binding modules, CtCBM6A (405 bp) and CtCBM6B (402 bp), at the C-terminus (Ahmed et al., 2013 ▶). CtGH43 was expressed, biochemically characterized and shown to be predominantly an α-l-arabinofuranosidase (Ahmed et al., 2013 ▶). The truncated CtGH43 shows a unique ability to catalyze both p-nitrophenol-α-l-arabinofuranoside (pNPAf) and p-nitrophenol-α-l-arabinopyranoside (pNPAp), although the catalytic efficiency was greater with pNPAf (Ahmed et al., 2013 ▶). Phylogenetic analysis of CtGH43 from C. thermocellum shows a close evolutionary relationship with GH43s linked to family 6 carbohydrate-binding modules (CBM6s) from C. cellulolyticum, C. papyrosolvens, C. cellulyticum and Acetovibrio cellulolyticus (Ahmed et al., 2012 ▶). CtGH43 shows maximum activity at 50°C and at pH 5.4 with pH stability in the range 5.0–6.2 (Das et al., 2012 ▶). Determining the three-dimensional structure of CtGH43 will further provide understanding of its activity towards different substrates. In the present study, recombinant CtGH43 was purified and crystallized. X-ray diffraction data were collected for preliminary structure analysis.

2. Materials and methods  

2.1. Overexpression and purification of CtGH43  

α-l-Arabinofuranosidase (CtGH43) from C. thermocellum was overexpressed in Escherichia coli BL21 pLysS (DE3) as described earlier (Das et al., 2012 ▶). The recombinant CtGH43 containing a His₆ tag was purified from the cell-free extract by immobilized metal-ion affinity chromatography (IMAC) using a Sepharose column (HiTrap Chelating, GE Healthcare) and was eluted using 50 mM HEPES buffer containing 300 mM imidazole, 1 M NaCl, 3 mM CaCl₂.2H₂O pH 7.5. Purified CtGH43 was buffer-exchanged into 50 mM HEPES buffer pH 7.5 containing 200 mM NaCl, 5 mM CaCl₂ using PD-10 columns and subjected to gel filtration on a HiLoad 16/60 Superdex-75 column at a flow rate of 1 ml min⁻¹. Purified CtGH43 was concentrated using a 10 kDa cutoff centrifugal concentrator (Millipore, USA) and washed three times with 0.5 mM CaCl₂. Its concentration was estimated using a molar extinction coefficient (∊) of 68 823 M ⁻¹ cm⁻¹. The recombinant CtGH43 contains an N-terminal His₆ tag (MGSSHHHHHHSSGLVPRGSHMAS), giving a total of 324 amino-acid residues with an approximate molecular mass of 35.86 kDa, as analyzed by SDS–PAGE (Das et al., 2012 ▶).

2.2. Crystallization of CtGH43  

Commercial screens such as Crystal Screen, Crystal Screen 2 and PEG/Ion from Hampton Research (California, USA), JCSG from Molecular Dimensions and an in-house-prepared sparse-matrix screen of 80 conditions were screened by the sitting-drop vapour-diffusion method for crystallization of CtGH43 using an Oryx8 nanodrop robotic dispensing system (Douglas Instruments). Drops consisting of 0.8 µl of either 60 or 120 mg ml⁻¹ CtGH43 solution and 0.8 µl reservoir solution were prepared at 19°C. Cube-shaped or brick-shaped crystals of CtGH43 grew after 10 d in different conditions in the initial screen as shown in Fig. 1 ▶. We also obtained cube-shaped crystals in just 2 M ammonium sulfate. These crystals were isomorphous, but diffracted less well than those from the condition given in Fig. 1 ▶(a).

Figure 1.

Crystals of CtGH43 produced by sitting-drop vapour diffusion grown with 120 mg ml⁻¹ protein under the following conditions: (a) 0.1 M sodium acetate pH 4.5, 2 M ammonium sulfate and (b) 0.1 M Tris pH 8.5, 20% PEG 2000 monomethyl ether, 0.2 M trimethyl N-oxide. The largest cube-shaped crystals (Fig. 1 ▶ a) are approximately 0.10 × 0.10 × 0.10 mm in size, whereas the brick-shaped crystal (Fig. 1 ▶ b) is ∼0.30 × 0.15 × 0.10 mm in size.

The crystals were cryocooled in liquid nitrogen after soaking in cryoprotectant [30%(v/v) glycerol added to the crystallization buffer] for a few seconds except for the crystal grown in 0.1 M Tris pH 8.5, 20% PEG 2000 monomethyl ether, 0.2 M trimethyl N-oxide, for which Paratone-N was used.

2.3. Data collection and processing of CtGH43 crystals  

The data from all the CtGH43 crystals obtained from different conditions were collected at the Diamond Light Source at the National Synchrotron Science Facility (Harwell, England) using a PILATUS 6M detector with the crystals cooled to −173°C using a Cryostream (Oxford Cryosystems). The data were processed by fast DP and xia2 (Winter, 2010 ▶) using the programs iMosflm (Battye et al., 2011 ▶) and AIMLESS (Evans, 2011 ▶) from the CCP4 suite (Winn et al., 2011 ▶) or XDS (Kabsch, 2010 ▶). For the cube-shaped crystals (Fig. 1 ▶ a), 120° of data were collected with a Δϕ of 0.2°. These gave diffraction data between 1.65 and 2.0 Å resolution; the data sets were highly isomorphous and the crystal belonged to the cubic space group P432. Thus, the data from the best diffracting crystal (1.65 Å resolution) were used to solve the CtGH43 structure. The Matthews coefficient of 3.17 ų Da⁻¹ (Matthews, 1968 ▶) indicates the presence of one molecule in the asymmetric unit with a solvent content of 61%.

For the brick-shaped crystal (Fig. 1 ▶ b), 400° of data were collected with a Δϕ of 0.2° in two passes, first to 1.5 Å resolution and then to 1.1 Å resolution (Fig. 2 ▶). This crystal belonged to the monoclinic space group P2₁. The Matthews coefficient of 2.0 ų Da⁻¹ (Matthews, 1968 ▶) also indicates the presence of one molecule in the asymmetric unit, with a solvent content of 39%. Data-collection statistics for both CtGH43 crystalline forms are given in Table 1 ▶. BALBES was used to carry out molecular replacement (Long et al., 2008 ▶). Five structures with a sequence identity of greater than 20% were found. The best solution was found using the GH43 arabinoxylan arabinofuranohydrolase from Bacillus subtilis (PDB entry 3c7e; Vandermarliere et al., 2009 ▶; sequence identity 41%). This gave a monomer in space group P432, with a final R factor and R _free of 35.9 and 37.4%, respectively, and a Q factor of 0.767 after REFMAC5 (Murshudov et al., 2011 ▶). Phaser (McCoy et al., 2007 ▶) was used to fit the PDB model output from BALBES into the monoclinic 1.1 Å resolution data, with a TFZ score of 27.5 and an LLG of 2062. As well as the difference in packing, there is a significant difference in some of the loops. Further structure refinement and analysis for both crystalline forms are ongoing.

Figure 2.

Representative diffraction pattern from the CtGH43 crystal grown in 0.1 M Tris pH 8.5, 20% PEG 2000 monomethyl ether, 0.2 M trimethyl N-oxide. The outer circle corresponds to 1.1 Å resolution.

Table 1. Data-collection statistics.

Values in parentheses are for the highest resolution shell.

Crystal	Cube-shaped	Brick-shaped
Beamline	I04, Diamond	I04, Diamond
Space group	P432	P21
Wavelength (Å)	0.9795	0.9795
Unit-cell parameters
a (Å)	140.0	45.43
b (Å)	140.0	67.70
c (Å)	140.0	50.99
β (°)	90	113.11
Resolution limits (Å)	57.15–1.65 (1.68–1.65)	27.45–1.10 (1.16–1.10)
Average mosaicity (°)	0.30	0.38
No. of observations	1175673 (31832)	707665 (75874)
No. of unique observations	56751 (2732)	104966 (13587)
Multiplicity	20.7 (11.7)	6.7 (5.6)
Completeness (%)	100 (100)	91.5 (81. 2)
〈I/σ(I)〉	20.0 (1.8)	13.4 (4.9)
CC1/2 †	0.99 (0.67)	0.997 (0.944)
R merge ‡ (%)	9.1 (115.4)	12.6 (6.3/46.6)
R p.i.m. § (%)	2.1 (36.5)	8.0 (3.5/30.4)

^†CC _1/2 is the half data-set correlation coefficient (Diederichs & Karplus, 2013 ▶).

^‡ R _merge = , where I_i(hkl) is the ith intensity measurement of reflection hkl, including symmetry-related reflections, and 〈I(hkl)〉 is its average.

^§ R _p.i.m. = , where 〈I(hkl)〉 is the average of symmetry-related observations of a unique reflection.