Crystallization and preliminary X-ray diffraction analysis of a new xyloglucanase from Xanthomonas campestris pv. campestris

X. campestris pv. campestris xyloglucanase was produced in E. coli using recombinant pSMT3 vector. Crystals were obtained and X-ray data sets were collected for both the apoenzyme and its complex with glucose.

Abstract

Xyloglucanases (Xghs) are important enzymes involved in xyloglucan modification and degradation. Xanthomonas campestris pv. campestris (Xcc) is a phytopathogenic bacterium which produces a large number of glycosyl hydrolases (GH), but has only one family 74 GH (Xcc-Xgh). This enzyme was overexpressed in Escherichia coli, purified and crystallized. Diffraction data sets were collected for the native enzyme and its complex with glucose to maximum resolutions of 2.0 and 2.1 Å, respectively. The data were indexed in a hexagonal crystal system with unit-cell parameters a = b = 153.4, c = 84.9 Å. As indicated by molecular-replacement solution, the crystals belonged to space group P6₁.

1. Introduction  

Exploration of new energy sources is a scientific and technologic challenge to modern societies. Renewable energy is an important alternative to fossil fuels (Goldemberg, 2008 ▶; Balat & Balat, 2009 ▶). Vegetal biomass is a renewable, sustainable and inexpensive material suitable for second-generation bioethanol production. The plant cell wall is constituted of cellulose fibres embedded in a polymer matrix and is composed of cellulose (∼15–40%), hemicellulose-pectin (∼30–40%) and lignin (∼20%) (Lynd et al., 2002 ▶; Himmel et al., 2007 ▶). Many microorganisms, such as bacteria and fungi, produce extracellular enzymes that are capable of degrading plant cell wall material by converting cellulose and hemicelluloses to fermentable sugars (Chundawat et al., 2011 ▶).

As part of the hemicellulose fraction of biomass, xyloglucan forms a complex network which functions as a bridge between cellulose microfibrils, giving rigidity and extension capacity to the cell walls of dicotyledons and some monocotyledons (Martinez-Fleites et al., 2006 ▶). This branched polymer is composed of a cellulose-like backbone of β-(1,4)-glucose with side chains of α-d-xylose, xylose-galactose or xylose-galactose-fucose and their diversity is plant dependent (Hayashi, 1989 ▶; Peng et al., 2012 ▶; Park & Cosgrove, 2012 ▶). Xyloglucan is a structural element that hinders access of enzymes to cellulose and thus biomass conversion by cellulases (Ding et al., 2012 ▶; Dixon, 2013 ▶). Thus, the efficient removal of xyloglucan is required for complete lignocellulosic biomass decomposition by the concerted action of cellulases and hemicellulases (Hayashi, 1989 ▶; Scheller & Ulvskov, 2010 ▶).

Xyloglucan-specific endo-β-1,4-glucanases or xyloglucanases (EC 3.2.1.151) are grouped into glycoside hydrolase families 5, 12, 16, 44 and 74 in the Carbohydrate-Active enZYmes Database (CAZy; http://www.cazy.org/; Cantarel et al., 2009 ▶; Gilbert et al., 2008 ▶; Damásio et al., 2012 ▶). Glycoside hydrolase family 74 (GH74) enzymes exhibit high specificity towards xyloglucan, except for Cel74 from Thermotoga maritima, which has been characterized as a cellobiohydrolase (Chhabra & Kelly, 2002 ▶).

It has been reported that endo-β-1,4-glucanases display hydrolytic activity towards xyloglucan and oligoxyloglucan (Yaoi & Mitsuishi, 2002 ▶; Bauer et al., 2005 ▶; Desmet et al., 2007 ▶; Irwin et al., 2003 ▶; Yaoi et al., 2004 ▶, 2009 ▶; Martinez-Fleites et al., 2006 ▶; Hasper et al., 2002 ▶; Ichinose et al., 2012 ▶; Enkhbaatar et al., 2012 ▶). From a structural point of view, this is a poorly studied group of enzymes, with only two crystal structures available in the Protein Data Bank (PDB): one from Clostridium thermocellum F7/YS (PDB entries 2cn2 and 2cn3; Martinez-Fleites et al., 2006 ▶) and one from Geotrichum sp. M128 (PDB entries 1sqj, 2ebs and 3aof; Yaoi et al., 2004 ▶, 2007 ▶, 2009 ▶).

Xanthomonas campestris pv. campestris (Xcc) is a Gram-negative bacterium that is able to synthesize a wide range of extracellular enzymes which hydrolyse different kinds of substrates. Analysing the genomic sequence data of Xcc strain ATCC33913 in CAZy (Cantarel et al., 2009 ▶), the uncharacterized gene xcc1752 (GenBank NP_637119.1) was identified. This gene was annotated as a putative cellulase (81.14 kDa) belonging to GH74.

We set out to determine the three-dimensional structure of this xyloglucanase from Xcc (Xcc-Xgh) in the hope that its structural characterization will contribute to the catalogue of GH74 structures and might provide insights into the molecular basis of the mode of action of bacterial xyloglucanases on xyloglucan polysaccharides.

2. Materials and methods  

2.1. Protein preparation  

The coding region of the Xcc-Xgh protein was amplified by polymerase chain reaction (PCR) from Xcc genomic DNA using the primer pair Xcc_1752F (5′-ACATATGGCCACGTCCGGGCCCTACCAGTG-3′) and Xcc_1752R (5′- AGAGCTCATGGCCACGTCCGGGCCCTACCAG-3′) containing NdeI and SacI restriction sites (shown in bold), respectively. The 2136 bp amplified fragment was introduced into pTZ57R/T vector (Fermentas, USA) and then subcloned into pSMT3 expression vector.

The recombinant plasmid pSMT3-Xcc-Xgh was transformed into Escherichia coli BL21 (DE3) cells and induced using 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 293 K for 24 h. Cells were harvested by centrifugation at 6000g and resuspended in lysis buffer (20 mM Tris–HCl pH 8.0, 300 mM NaCl). Cells were lysed by sonication and the supernatant was centrifuged at 1800g to remove cell debris. The supernatant was subjected to affinity purification using Ni–NTA resin (Qiagen) equilibrated with 20 mM Tris–HCl pH 8.0, 150 mM NaCl, 20 mM imidazole buffer. The Xcc-Xgh protein eluted at approximately 300 mM imidazole in a 50–300 mM imidazole gradient. The protein was dialysed against buffer consisting of 20 mM Tris–HCl pH 8.0, 150 mM NaCl and then subjected to ubiquitin-like protein-specific protease 1 (ULP1) digestion at 277 K for 24 h to remove the His tag. The resulting mixture was again loaded onto Ni–NTA resin and untagged Xcc-Xgh was collected in the flowthrough. Fractions containing the target protein were loaded onto a HiLoad Superdex 75 16/60 size-exclusion chromatography column (GE Healthcare) pre-equilibrated with 20 mM Tris–HCl pH 8.0, 150 mM NaCl using an ÄKTApurifier 10 system (GE Healthcare Life Sciences) to remove aggregates and impurities from the protein of interest.

The final protein sample has >95% purity as assessed by 15% SDS–PAGE stained with Coomassie Brilliant Blue (Laemmli, 1970 ▶). The purified Xcc-Xgh was concentrated to 42 mg ml⁻¹ in 20 mM Tris–HCl pH 8.0, 50 mM NaCl for crystallization assays. The protein concentration was determined by measuring absorption at 280 nm using a NanoDrop spectrophotometer (Thermo Scientific).

2.2. Crystallization and data collection  

Initial crystallization screens were performed using a Honeybee automated robotic system at the Laboratório Robotizado de Cristalização de Proteínas (Robolab-LNLS, Campinas, Brazil) using 480 different reservoir conditions from kits from Hampton Research (Laguna Niguel, California, USA) and Emerald BioSystems (Bainbridge Island, Washington, USA), including Crystal Screen, Crystal Screen 2, Wizard I and II, and SaltRx. The sitting-drop vapour-diffusion method was used, in which 1 µl Xcc-Xgh solution (in 20 mM Tris–HCl pH 8.0, 50 mM NaCl) was mixed with 1 µl reservoir solution in 96-well Cryschem Plates (Hampton Research). For data collection, a single apoprotein crystal was transferred to a cryoprotectant solution containing 15%(v/v) ethylene glycol and was flash-cooled at 100 K. A diffraction data set for Xcc-Xgh complexed with glucose was collected from a crystal soaked in cryoprotectant solution containing 1 M glucose.

Diffraction data were collected on the MX2 beamline of the Brazilian Synchrotron Light Laboratory, Campinas, Brazil, using a wavelength of 1.48 Å (Guimarães et al., 2009 ▶). A total of 360 images covering an oscillation range of 180° were collected using a MAR Mosaic 225 mm CCD detector (MAR Research). The diffraction images were processed using XDS (Kabsch, 2010 ▶) and reduced with SCALA from CCP4 (Evans, 2006 ▶, 2011 ▶; Winn et al., 2011 ▶). POINTLESS (Evans, 2006 ▶) was used to examine the symmetry of the diffraction pattern and score the possible crystallographic symmetry and the phenix.xtriage module of PHENIX (Adams et al., 2010 ▶) was used to analyse the collected data for twinning.

3. Results and discussion  

The Xcc-Xgh protein was submitted to several crystallization trials and small crystals were obtained by a sitting-drop method in a condition consisting of 100 mM sodium cacodylate buffer pH 6.5, 200 mM calcium acetate, 18%(w/v) polyethylene glycol 8000; however, the macroscopic form of the crystals resembled a shower of needles (Fig. 1 ▶ a). In order to obtain diffraction-quality crystals, the condition of initial crystallization was exhaustively optimized and the best crystals with a typical size of 0.2 mm in the largest dimension were obtained in 100 mM sodium cacodylate buffer pH 6.5, 18% PEG 8000, 200 mM calcium acetate at 291 K and grew in 1 d (Fig. 1 ▶ b).

Figure 1.

Crystals of Xcc-Xgh obtained during initial screening (a) and after optimization of crystallization conditions (b).

A diffraction data set was collected to 2.0 Å resolution at 100 K from a cryoprotected apoprotein crystal (Table 1 ▶). A second diffraction data set for the enzyme in complex with glucose was collected to 2.1 Å resolution. The glucose-soaked crystal has slightly different unit-cell parameters (Table 1 ▶) compared wth the uncomplexed crystal, suggesting an impact of glucose on the crystal lattice. Assuming the presence of one molecule in the asymmetric unit, the solvent content of the crystals was estimated to be ∼50.0% based on the Matthews coefficient (Matthews, 1968 ▶). The data scaling suggested the point group P6 and systematic absences indicated the enantiomorphic space groups P6₁ or P6₅. This ambiguity was solved by the molecular-replacement process, which was conducted using the crystallographic structure of the homologous enzyme from C. thermocellum F7/YS (PDB entry 2cn2; Martinez-Fleites et al., 2006 ▶) and clearly revealed P6₁ as the correct space group. Refinement of the structures is in progress.

Table 1. Crystallographic data and refinement statistics.

Values in parentheses are for the outermost resolution shell.

	Apoenzyme	Complex with glucose
Source	MX2, LNLS	MX2, LNLS
Wavelength (Å)	1.4586	1.4586
Space group	P61	P61
Unit-cell parameters (Å)
a = b	153.43	150.53
c	84.92	84.20
Resolution (Å)	19.7–2.00 (2.30–2.00)	42.5–2.10 (2.40–2.10)
No. of reflections	837436	149921
No. of unique reflections	76752	55997
R merge † (%)	6.5 (28.6)	7.1 (72.4)
〈I/σ(I)〉	26.86 (9.08)	29.1 (1.5)
Completeness (%)	99.8 (100)	99.3 (100)
Multiplicity	10.9 (10.7)	2.7 (2.6)

^† R _merge = .