Expression at 279 K, purification, crystallization and preliminary X-ray crystallographic analysis of a novel cold-active β-1,4-d-mannanase from the Antarctic springtail Cryptopygus antarcticus

A cold-active β-1,4-d-mannanase from the Antarctic springtail C. antarcticus (CaMan) has been expressed, purified and crystallized. The CaMan crystal belonged to space group P2₁2₁2₁, with unit-cell parameters a = 73.40, b = 83.81, c = 163.55 Å, and diffracted to 2.35 Å resolution. A 2.40 Å resolution data set was also collected from a CaMan–mannopentaose complex crystal.

Abstract

The CaMan gene product from Cryptopygus antarcticus, which belongs to the glycoside hydrolase family 5 type β-1,4-d-mannanases, has been crystallized using a precipitant solution consisting of 0.1 M Tris–HCl pH 8.5, 25%(w/v) polyethylene glycol 3350 by the microbatch crystallization method at 295 K. The CaMan protein crystal belonged to space group P2₁2₁2₁, with unit-cell parameters a = 73.40, b = 83.81, c = 163.55 Å. Assuming the presence of two molecules in the asymmetric unit, the solvent content was estimated to be about 61.29%. CaMan–mannopentaose (M5) complex crystals that were isomorphous to the CaMan crystals were obtained using the same mother liquor containing 1 mM M5.

1. Introduction  

Mannan is a major component of hemicelluloses in plants, plant seeds and some algae. There are four kinds of mannan: two linear mannans (pure mannan and glucomannan) and two branched mannans (galactomannan and galactoglucomannan) (Petkowicz et al., 2001 ▶; Moreira & Filho, 2008 ▶; Tailford et al., 2009 ▶). Each of these polysaccharides presents a β-1,4-linked backbone containing mannoses (pure mannan) or a combination of glucoses and mannoses (glucomannan) (Liepman et al., 2007 ▶). In addition, the mannan backbone can be branched with side chains of α-1,6-linked galactose residues (galactomannan or galactoglucomannan). In some cases, such as acemannans, the mannose and glucose residues in the backbone are acetylated at C2 or C3 (Jansson et al., 1975 ▶). The degradation of mannans requires the concerted action of a variety of hydrolytic enzymes owing to their structural complexity. β-Mannanases (β-1,4-d-mannan mannohydrolases; EC 3.2.1.78), β-mannosidases (β-1,4-d-mannopyranoside hydrolases; EC 3.2.1.25) and β-glucosidases (β-1,4-d-glucoside glucohydrolases; EC 3.2.1.21) degrade the β-1,4-linkage in the mannan backbone. In addition, acetyl mannan esterases (EC 3.1.1.6) and α-galactosidases (α-1,6-d-galactoside galactohydrolases; EC 3.2.1.22) are required to remove the acetyl group and side-chain substituents, respectively, which are attached at various points on the mannan structures (Moreira & Filho, 2008 ▶). These enzymes are important for microorganisms and animals that utilize various plant β-mannans as food sources. They have also gained industrial interest owing to their ability to modify and degrade mannan-containing polysaccharides in paper production and the food and feed industries (Marga et al., 1996 ▶).

Based on their primary sequences, β-mannanases are grouped mainly into glycoside hydrolase (GH) families 5 and 26, both of which belong to the largest clan GH-A featuring the TIM (triosephos­phate isomerase) (β/α)₈-barrel fold (Zhao et al., 2009 ▶; Gilbert, 2010 ▶). The crystal structures of β-mannanases belonging to both GH families from a wide range of bacteria, fungi, plant and animals have been studied (Hogg et al., 2001 ▶; Le Nours et al., 2005 ▶; Cartmell et al., 2008 ▶; Tailford et al., 2009 ▶; Songsiriritthigul et al., 2011 ▶; Zhao et al., 2011 ▶; Hilge et al., 1998 ▶; Sabini et al., 2000 ▶; Bourgault et al., 2005 ▶; Larsson et al., 2006 ▶; Yan et al., 2008 ▶; Zhang et al., 2008 ▶; Mizutani et al., 2012 ▶; Gonçalves et al., 2012 ▶; Couturier et al., 2013 ▶), revealing an open active-site cleft with at least four subsites and two catalytic glutamates (nucleophiles and acid/base). Ligand-complex structures indicate that GH5 and GH26 β-mannanases have aromatic platforms distributed in the active-site cleft that interact with the hydrophobic α-faces of substrate sugar rings (Gilbert et al., 2008 ▶).

The freezing-intolerant Antarctic springtail Cryptopygus antarcticus Willem (Collembola, Isotomidae) is the most abundant and widespread terrestrial micro-arthropod in the maritime Antarctic region. The adult reaches approximately 1.2 mm in body length with a live weight of 55–78 µg and contains 69–85% water. This organism feeds on fungi, unicellular algae and detritus. Therefore, it is thought that C. antarcticus contains various kinds of cold-active enzymes that hydrolyse carbohydrates. From an expressed sequence tags (ESTs) library of C. antarcticus, we identified the β-1,4-d-mannanase gene (CaMan) that belongs to the GH5 family. CaMan is a 382-residue protein with a putative signal peptide and exhibits high specific activity towards locust bean gum at an optimal temperature of 303 K and an optimal pH of 3.5 (Song et al., 2008 ▶). Its optimal temperature is the lowest among those of the known mannanases and the optimal pH is also the lowest except for those of the Sclerotium rolfsii and Aspergillus sulfurous enzymes (pH 2.9 and pH 2.4, respectively; Gübitz et al., 1996 ▶; Chen et al., 2007 ▶). Even at 273–278 K, CaMan retains 20–40% of its maximum activity. It also shows typical features of a cold-active enzyme, with a high frequency of polar residues such as Asn, Gln and Ser and a low frequency of hydrophobic residues as well as a low ratio of Arg/(Arg+Lys) compared with the mesophilic β-­mannanases (Russell, 2000 ▶; Margesin & Schinner, 1999 ▶). Here, we report the overexpression, purification, crystallization and preliminary X-ray crystallographic analyses of CaMan, which is the first β-­1,4-d-mannanase from an arthropod and from a multi-cellular animal other than molluscs, and its complex with mannopentaose (M5).

2. Materials and methods  

2.1. Expression at 279 K and purification of the CaMan protein  

The CaMan gene (GenBank accession No. ABV68808.1) without the signal peptide-coding region was amplified by the polymerase chain reaction using the EST library of C. antarcticus as a template (Song et al., 2008 ▶). The gene was inserted downstream of the T7 promoter of the expression plasmid pET-24a (Novagen, Madison, Wisconsin, USA) and the resulting construct expressed residues 22–382 of the CaMan protein with a C-terminal noncleavable His₆ tag (LEHHHHHH). After verifying the DNA sequence, plasmid DNA was transformed into Escherichia coli strain Rosetta-gami2 (DE3) (Stratagene, La Jolla, California, USA). The cells were grown to an OD₆₀₀ of approximately 0.5 in Luria–Bertani medium containing 50 µg ml⁻¹ kanamycin (Duchefa) at 310 K and expression was induced by 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG; Duchefa). To solubilize the active CaMan protein, we introduced the extremely low temperature induction system (Song et al., 2012 ▶). After 7 d induction at 279 K, the cells were harvested and resuspended in 20 mM Tris–HCl pH 7.4 containing 5 mM imidazole. The cells were disrupted by sonication and the cell debris was discarded by centrifugation at 20 000g for 30 min at 277 K. The resulting supernatant was loaded onto TALON metal-affinity resin (Clontech, USA). The column was washed with buffer consisting of 20 mM Tris–HCl pH 7.4, 20 mM imidazole. The CaMan protein was eluted with the same buffer containing 300 mM imidazole. The protein fraction partially purified by the TALON resin was dialysed into 20 mM Tris–HCl pH 7.4, 1 mM dithiothreitol (DTT) and loaded onto a Superdex 75 HR 16/60 column (GE Healthcare) pre-equilibrated with buffer consisting of 20 mM Tris–HCl pH 7.4, 300 mM NaCl, 1 mM DTT (buffer A). The CaMan protein eluted at ∼70.1 min at a flow rate of 1.5 ml min⁻¹. The purified CaMan in buffer A was concentrated to approximately 24 mg ml⁻¹ for crystallization.

2.2. Crystallization and X-ray data collection  

The CaMan protein was crystallized and optimized by the micro-batch crystallization method at 295 K. Small drops composed of 1 µl protein solution and an equal volume of crystallization reagent were pipetted under a layer of a 1:1 mixture of silicon oil and paraffin oil in 96-well IMPACT plates (Greiner Bio-One) or 72-well HLA plates (Nunc). Screening for crystallization conditions was performed with all available screening kits from Hampton Research, Axygen Bio­sciences and Emerald BioSystems. Initial crystals (Fig. 1 ▶) were grown in a precipitant solution consisting of 0.1 M Tris–HCl pH 8.5, 25%(w/v) polyethylene glycol (PEG) 3350 (condition No. 45 of Index from Hampton Research). The rod-shaped crystals were produced in 3 d. To obtain the CaMan–M5 complex crystal, CaMan protein was incubated with 1 mM M5 and crystallized under the same conditions as the native CaMan crystal.

Figure 1.

Crystal of the CaMan protein.

The CaMan and CaMan–M5 crystals were mounted using a nylon loop (50 µm Mounted CryoLoop from Hampton Research) for data collection and were cooled to 100 K using a Cryostream cooler (Oxford Cryosystems) after slowly concentrating a drop of the mother liquor by leaving the drop open to air and allowing the drop to slowly evaporate for 8 min. A 2.35 Å resolution data set for CaMan was collected using an ADSC Quantum 315r CCD on beamline BL-17A of the Photon Factory, Tsukuba, Japan. The exposure time to the synchrotron radiation was 5 s. A total of 191 frames of 1° oscillation were measured with the crystal-to-detector distance set to 285 mm. A 2.40 Å resolution data set for the CaMan–M5 complex was collected using an ADSC Quantum 210 CCD on beamline BL-4A at Pohang Light Source, Pohang, Republic of Korea. The exposure time to the synchrotron radiation was 4 s. A total of 240 frames of 1° oscillation were measured with the crystal-to-detector distance set to 350 mm. Diffraction data were processed and scaled using DENZO and SCALEPACK from the HKL-2000 program suite (Otwinowski & Minor, 1997 ▶). The data-collection statistics are shown in Table 1 ▶.

Table 1. Data-collection and processing statistics.

Values in parentheses are for the outermost resolution shell.

	CaMan	CaMan–M5
Beamline	BL-17A, PF	BL-4A, PAL
Space group	P212121	P212121
Unit-cell parameters (Å)	a = 73.40, b = 83.81, c = 163.55	a = 74.01, b = 82.31, c = 164.26
Wavelength (Å)	0.96416	1.0000
Resolution (Å)	50.00–2.35 (2.39–2.35)	50.00–2.40 (2.44–2.40)
Completeness (>0σ) (%)	96.3 (88.9)	97.7 (96.3)
R merge † (%)	5.0 (18.4)	9.6 (30.7)
Average I/σ(I)	24.5 (3.1)	11.7 (1.9)
No. of observations	232051	214319
No. of unique reflections	41345 (1891)	39246 (1907)
Multiplicity	5.6 (4.1)	5.5 (3.5)

^† R _merge = , where I_i(hkl) is the intensity of observed reflection hkl and 〈I(hkl)〉 is the mean intensity of replicate measurements.

3. Results and discussion  

The CaMan protein formed inclusion bodies when overexpressed at 288–310 K. This insolubility problem was overcome by simply lowering the temperature to 279 K as previously described (Song et al., 2012 ▶). Recombinant CaMan protein expressed at this extremely low temperature exhibited mannanase activity (Song et al., 2012 ▶) and was successfully crystallized. CaMan shows the highest specific activity towards locust bean gum and is also active towards konjac and β-1,4-mannan (Song et al., 2008 ▶). It also digests mannotetraose (M4) and mannopentaose (M5) to give mannobiose (M2) and mannotriose (M3) with a little production of mannose (M1). However, CaMan hardly degraded M2 and M3 (Song et al., 2008 ▶). To elucidate the structural basis for its catalytic features, we tried to obtain native CaMan and CaMan–M5 complex crystals. The CaMan crystal belonged to the primitive orthorhombic space group P2₁2₁2₁, with unit-cell parameters a = 73.40, b = 83.81, c = 163.55 Å. The crystal volume per unit molecular weight (V _M) was about 3.20 ų Da⁻¹ with a solvent content of 61.29% by volume (Matthews, 1968 ▶) when the asymmetric unit was assumed to contain two molecules. The CaMan–M5 complex crystal belonged to the primitive orthorhombic space group P2₁2₁2₁, with unit-cell parameters a = 74.01, b = 82.31, c = 164.26 Å. The crystal volume per unit molecular weight (V _M) was about 3.18 ų Da⁻¹ with a solvent content of 61.29% by volume when the asymmetric unit was assumed to contain two molecules (Table 1 ▶).

CaMan shows 41, 40, 41 and 40% sequence identity to other invertebrate β-mannanases from the abalone Haliotis discus hannai (HdMan), the common sea hare Aplysia kurodai (AkMan), the freshwater snail Biomphalaria glabrata (BgMan) and the blue mussel Mytilus edulis (MeMan5A), respectively. The primary sequence of CaMan aligns well with these β-mannanases, all of which belong to GH5 subfamily 10. However, this enzyme shows low sequence identities ranging from 13.8 to 8% to other β-mannanases from plants, fungi and bacteria. The sequence alignment reveals that CaMan has an extended loop region (349–352; Fig. 2 ▶). Hitherto, two crystal structures of invertebrate mollusc β-mannanases [AkMan (PDB entry 3vup; Mizutani et al., 2012 ▶) and MeMan5A (PDB entry 2c0h; Larsson et al., 2006 ▶)] have been available. According to these structures, the extended loop of CaMan is highly likely to be located at the entrance of the active-site cleft. Structural or sequential modifications of loops in the active site have been reported to affect the discrimination of mannan substrates (Gonçalves et al., 2012 ▶; Cartmell et al., 2008 ▶). From this perspective, the extended loop of CaMan in the active-site cleft is likely to be critical to its catalytic features. Molecular replacement was performed with MOLREP (Vagin & Teplyakov, 2010 ▶) using the structure of MeMan5A (Larsson et al., 2006 ▶) as a search model. Using the apo CaMan data set, the rotation search, which was performed within the resolution range 45.46–2.73 Å, yielded two top solutions with Rf/σ of 9.22 and 7.90 (in contrast to 5.85 for the third solution), indicating the presence of two monomers per asymmetric unit. The best solution after the translation function had an R factor of 65.60%. Restrained refinement of this solution with REFMAC5 (Murshudov et al., 2011 ▶) significantly lowered the R factors to R = 37.93% and R _free = 44.12% with a figure of merit of 0.57 in the 45.80–2.35 Å resolution range. Using the CaMan–M5 data set, the rotation search, which was performed within the resolution range 35.97–2.56 Å, yielded two top solutions with Rf/σ of 8.08 and 6.95 (in contrast to 4.27 for the third solution), indicating the presence of two monomers per asymmetric unit. The best solution after the translation function had an R factor of 61.60%. Restrained refinement of this solution with REFMAC5 (Murshudov et al., 2011 ▶) lowered the R factors to R = 38.76% and R _free = 44.37% with a figure of merit of 0.57 in the 35.99–2.40 Å resolution range. From difference Fourier maps of the CaMan–M5 complex, we could identify M5 in the active site. To our knowledge, this is the first structure of a GH5 β-mannanase in complex with M5 and thus will provide detailed insights into the substrate-recognition mechanism and enzymatic features of CaMan as well as the functional implications of the extended loop. Manual model improvement and refinement are now in progress.

Figure 2.

Sequence alignment of C. antarcticus β-mannanase (CaMan) with those of H. discus hannai (HdMan), A. kurodai (AkMan), B. glabrata (BgMan) and M. edulis (MeMan5A). Amino acids highlighted with triangles indicate the catalytic glutamates (black), active-site residues (blue), conserved glycines (green), other highly conserved residues (orange) and GH5 subfamily 10-specific tryptophans (purple). CaMan has an extended loop region (349–352; black box).