Overproduction, purification, crystallization and preliminary X-ray characterization of a novel carbohydrate-binding module of endoglucanase Cel5A from Eubacterium cellulosolvens

The novel N-terminal carbohydrate-binding module of the family 5 glycoside hydrolase endoglucanase Cel5A from E. cellulosolvens has been crystallized and MAD data have been collected to 1.75 Å resolution.

Abstract

The anaerobic cellulolytic rumen bacterium Eubacterium cellulosolvens produces a large array of cellulases and hemicellulases that are responsible for the hydrolysis of plant cell-wall polysaccharides. One of these enzymes, endoglucanase Cel5A, comprises two tandemly repeated novel carbohydrate-binding modules (CBMs) and two catalytic domains belonging to glycoside hydrolase family 5 joined by flexible linker sequences. The novel CBM located at the N-terminus of the endoglucanase has been crystallized. The crystals belonged to the hexagonal space group P6₁22 and contained a single molecule in the asymmetric unit. The structure of the l-selenomethionine derivative has been solved by a MAD experiment on crystals that diffracted to 1.75 Å resolution.

1. Introduction

In animals, the breakdown of plant feedstocks, particularly cellulose and other recalcitrant carbohydrates, occurs through fermentation by microbes in the rumen or the hindgut (Bryant et al., 1958 ▶; Flint & Bayer, 2008 ▶; Prins et al., 1972 ▶). Recalcitrant polysaccharides found in the plant cell wall pose numerous obstacles to hydrolysis through their development of complex chemical structures and also by becoming inaccessible to biocatalysts owing to the intricacy of their macromolecular structure. Thus, anaerobic bacteria and fungi in the rumen have developed a wide array of multi-modular cellulases and hemicellulases that act individually and as organized cellulosomes (an extremely complex and dynamic extracellular macromolecular assembly) for the hydrolysis of plant cell-wall polysaccharides to soluble sugars (for reviews, see Bayer et al., 2004 ▶; Fontes & Gilbert, 2010 ▶). Glycoside hydrolases (GH) form one of the most widespread groups of enzymes which hydrolyze the glycosidic bond between polysaccharides, with more than 120 distinct families classified to date (see the CAZy website; http://www.cazy.org; Cantarel et al., 2009 ▶). The Eubacterium cellulosolvens endoglucanase Cel5A (EcCel5A) has been sequenced and characterized (Yoda et al., 2005 ▶). It is a 1148-amino-acid protein comprising a tandem repeat of an unclassified carbohydrate-binding module (here termed CBM-AL1/2) linked to a glycoside family 5 catalytic module (GH5) and a PT-rich module (PT). Thus, the architectural arrangement of Cel5A is N–CBM-AL1–GH5-1–PT1–CBM-AL2–GH5-2–PT2–C flanked by an N-terminal signal peptide and a C-terminal tail of unknown function. The two CBMs and GH5s show sequence identities of 73 and 94%, respectively, to each other. The enzyme has been shown to have activity towards a variety of cellulosic polysaccharides, including carboxy­methyl cellulose, lichenan, acid-swollen cellulose and oat-spelt xylan (Yoda et al., 2005 ▶). The catalytic domains of Cel5A display moderate (20–40%) sequence identity to other known GH5s (see CAZy database) and are thus expected to present the (β/α)₈ TIM-barrel fold common to the GH-A clan (Henrissat, 1998 ▶). However, a BLAST search (Altschul et al., 1990 ▶) showed that there are no structural homologues of CBM-AL1. In fact, to date only two other endo­glucanases, one from Cellulosilyticum ruminicola (Cai et al., 2010 ▶) and one from Clostridium lentocellum (Lucas et al., 2010 ▶), have been found to possess this CBM module along with a GH5 catalytic module. In order to gain insight into the structural properties that govern ligand recognition by this novel CBM, we aimed to determine the crystal structure of E. cellulosolvens CBM-AL1. In the present communication, we describe the overproduction, purification, crystallization and preliminary X-ray analysis of the EcCel5A N-terminal CBM-AL1 module.

2. Materials and methods

2.1. Protein production and purification

EcCel5A is a modular enzyme containing an N-terminal CBM followed by a GH5 catalytic domain. The two domains are duplicated in tandem and the enzyme contains an additional C-terminal domain of unknown function. The gene encoding the N-terminal CBM of EcCel5A (residues 37–170) was synthesized (NZYTech Ltd, Portugal) with codon usage optimized for expression in Escherichia coli. The synthesized gene contained engineered NheI and XhoI recognition sequences at the 5′ and 3′ ends, respectively, which were used for subsequent subcloning into pET-28a (Novagen), generating pAL1 which encodes CBM-AL1. CBM-AL1 contains an N-terminal His₆ tag. E. coli Tuner DE3 cells harbouring pAL1 were cultured in Luria–Bertani broth at 310 K to mid-exponential phase (A _600nm = 0.6) and recombinant protein overproduction was induced by the addition of 0.2 mM isopropyl β-d-1-thiogalactopyranoside and incubation for a further 16 h at 292 K. The His₆-tagged recombinant protein was purified from cell-free extracts by immobilized metal-ion affinity chromatography (IMAC) as described previously (Najmudin et al., 2005 ▶). Purified CBM-AL1 was buffer-exchanged into 50 mM HEPES–HCl pH 7.5, 200 mM NaCl, 5 mM CaCl₂ and then subjected to gel filtration using a HiLoad 16/60 Superdex 75 column (GE Healthcare) at a flow rate of 1 ml min⁻¹. Preparation of E. coli to generate selenomethionylated CBM-AL1 was performed as described in Carvalho et al. (2004 ▶) and the protein was purified using the same procedures as employed for the native CBM. Purified CBM-AL1 (Fig. 1 ▶) was concentrated using an Amicon 10 kDa molecular-mass centrifugal concentrator and washed three times with 5 mM DTT (for the SeMet protein) or water (for native CBM).

Figure 1.

A Coomassie Brilliant Blue-stained 14% PAGE gel evaluation of protein purity. Lane 1, molecular-mass markers (kDa); lane 2, CBM-AL1.

2.2. Crystallization

Crystallization conditions were screened by the hanging-drop vapour-diffusion method using Crystal Screen, Crystal Screen 2 and PEG/Ion Screen (Hampton Research). Drops consisting of 1 µl 80 mg ml⁻¹ CBM-AL1 solution and 1 µl reservoir solution were prepared at 292 K. Crystals (maximum dimension of ∼50 µm) grew within 3 d in the following two conditions: (i) 0.2 M Li₂SO₄, 25% polyethylene glycol monomethyl ether 2000 (PEG 2K MME) and (ii) 0.2 M ammonium sulfate, 0.1 M sodium acetate pH 4.6, 30%(m/v) PEG 2K MME. Crystals of l-selenomethionine-containing protein were also obtained by vapour diffusion using the hanging-drop method with equal volumes (1 µl) of protein solution (80 mg ml⁻¹ in 5 mM DTT) and reservoir solution from a fine screen based around the above two successful conditions for the native crystals. The best crystals (maximum dimension of ∼100 µm) grew over a few days in 0.20–0.23 M ammonium sulfate, 0.1 M sodium acetate pH 4.6, 26%(m/v) PEG 2K MME in clusters (Fig. 2 ▶). The crystals were cryocooled in liquid nitrogen after soaking in cryoprotectant [30%(v/v) glycerol added to the crystallization buffer] for a few seconds.

Figure 2.

Crystals of CBM-AL1 obtained by hanging-drop vapour diffusion in the presence of 0.23 M ammonium sulfate, 0.1 M sodium acetate pH 4.6, 26%(m/v) PEG 2K MME. The largest crystals are approximately 120 × 30 × 30 µm in size. Crystals were prised apart by gentle prodding with a needle before choosing the best one for data collection.

2.3. Data collection and processing

MAD data were collected for selenomethionylated CBM-AL1 on beamline ID23-1 at the ESRF (Grenoble, France) using a Quantum 315r charge-coupled device detector (ADSC, USA) from a crystal cooled to 100 K using a Cryostream (Oxford Cryosystems Ltd.) (Table 1 ▶ and Fig. 3 ▶). All data sets were processed using the programs iMOSFLM (Leslie, 1992 ▶) and SCALA (Evans, 2006 ▶) from the CCP4 suite (Collaborative Computational Project, Number 4, 1994 ▶). The crystals belonged to the hexagonal system with either the P6₁22 or P6₅22 space-group enantiomorph as determined by POINTLESS (Evans, 2006 ▶). The Matthews coefficient (V _M = 2.11 ų Da⁻¹) suggested the presence of one molecule in the asymmetric unit and a solvent content of 42% (Matthews, 1968 ▶). The SeMet crystal diffracted to a resolution of 1.75 Å. Data were collected at 0.97934 Å (inflection point, f′ = −9.72, f′′ = 3.43), 0.97930 Å (peak, f′ = −8.10, f′′ = 5.38) and 0.97560 Å (remote). The crystal was fragile and suffered radiation damage after peak-data collection. Thus, the structure was solved by the SAS protocol of Auto-Rickshaw using only the peak data with the EMBL Hamburg automated crystal structure determination platform (Panjikar et al., 2005 ▶). The input diffraction data were processed and converted for use in Auto-Rickshaw using programs from the CCP4 suite (Collaborative Computational Project, Number 4, 1994 ▶). F _A values were calculated using the program SHELXC (Sheldrick, 2008 ▶). Based on an initial analysis of the data, the maximum resolution for substructure determination and initial phase calculation was set to 1.75 Å. Both of the heavy atoms requested were found using the program SHELXD (Sheldrick, 2008 ▶) and SHELXE gave better statistics for the P6₁22 space group. 94% of the model was built using the program ARP/wARP (Perrakis et al., 1999 ▶; Morris et al., 2004 ▶).

Table 1. Data-collection statistics.

Values in parentheses are for the lowest/highest resolution shells.

Data set	Peak	Inflection point	Remote
Wavelength (Å)	0.9793	0.97934	0.9769
Unit-cell parameters
a = b (Å)	47.86	48.04	48.21
c (Å)	191.60	191.97	192.34
Resolution limits (Å)	63.87–1.75 (63.87–5.53/1.84–1.75)	63.99–1.75 (63.99–5.53/1.84–1.75)	64.11–1.75 (64.11–5.53/1.84–1.75)
No. of observations	76770 (2783/6703)	77755 (2577/7493)	76501 (2588/6260)
No. of unique observations	13971 (558/1889)	14089 (526/1959)	14094 (530/1836)
Multiplicity	5.5 (5.0/3.5)	5.5 (4.9/3.8)	5.4 (4.9/3.4)
Completeness (%)	99.1 (97.6/94.8)	99.4 (93.2/98.2)	98.5 (92.9/91.6)
〈I/σ(I)〉	10.4 (22.5/1.7)	9.6 (19.9/1.7)	15.1 (1.3)
Rmerge†	6.8 (3.7/31.8)	7.5 (3.7/32.0)	7.1 (3.8/31.4)

^† R _merge = , where I_i(hkl) is the intensity of the measurement of reflection hkl and 〈I(hkl)〉 is the mean value of I_i(hkl) for all i measurements.

Figure 3.

Representative diffraction pattern of an SeMet CBM-AL1 crystal (the outer circle corresponds to 1.75 Å resolution).

Structure completion and analysis are ongoing.