Crystallization and preliminary crystallographic analysis of dextranase from Streptococcus mutans

S. mutans dextranase was crystallized by the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 1.6 Å and belonged to space group P2₁.

Abstract

Streptococcus mutans dextranase hydrolyzes the internal α-1,6-linkages of dextran and belongs to glycoside hydrolase family 66. An N- and C-terminal deletion mutant of S. mutans dextranase was crystallized by the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 1.6 Å and belonged to space group P2₁, with unit-cell parameters a = 53.2, b = 89.7, c = 63.3 Å, β = 102.3°. Assuming that the asymmetric unit of the crystal contained one molecule, the Matthews coefficient was calculated to be 4.07 ų Da⁻¹; assuming the presence of two molecules in the asymmetric unit it was calculated to be 2.03 ų Da⁻¹.

1. Introduction

Dextranases (EC 3.2.1.11) hydrolyze the internal α-1,6-linkages of dextran in an endo reaction mode, producing isomaltooligosaccharides of various sizes. Based on the similarities in their amino-acid sequences, dextranases are mainly classified into glycoside hydrolase family (GH) 49 and GH 66 in the CAZY database (Henrissat & Bairoch, 1996 ▶; Cantarel et al., 2009 ▶; http://www.cazy.org/). Dextran­ases belonging to GH 49 are found in bacteria and fungi, and a crystallographic study has revealed that the catalytic domain of Dex49A from Penicillium minioluteum is composed of a right-handed parallel β-helix (Larsson et al., 2003 ▶). GH 66 enzymes consist of dextranases and cycloisomaltooligosaccharide glucanotransferases (CITases; EC 2.4.1.248) and these enzymes are mainly found in bacteria. Both enzymes utilize dextran as a substrate, but CITases synthesize cycloisomaltooligosaccharide (a cyclic saccharide linked by α-1,6-glucosyl units) from dextran by intramolecular transglycos­ylation (Oguma et al., 1993 ▶, 1994 ▶; Funane et al., 2011 ▶). Amino-acid sequence comparison among GH 66 enzymes revealed that they are composed of four regions: an N-terminal variable region, a conserved region, a glucan-binding region and a C-terminal variable region (Igarashi et al., 2002 ▶, 2004 ▶; Morisaki et al., 2002 ▶), together with a CITase-specific region which is only observed in CITases (Funane et al., 2011 ▶). Although no three-dimensional structures of GH 66 enzymes have been determined, the similarity of the amino-acid sequence of the conserved region to members of GH 13, GH 27, GH 31 and GH 36 implies that GH 66 enzymes share a common (β/α)₈-barrel core with these families (Rigden, 2002 ▶). GH 49 enzymes act with an inverting catalytic mechanism, but GH 66 enzymes, as well as GH 13, GH 27, GH 31 and GH 36 enzymes, are known to employ a retaining mechanism.

Dextranase from Streptococcus mutans (SmDex), a bacterium found in human dental plaque, is a well studied enzyme in GH 66. S. mutans synthesizes extracellular α-glucans from sucrose using glucosyltransferases and hydrolyzes the α-1,6-linkages in the glucans using SmDex to produce isomaltooligosaccharides for metabolic utilization (Walker et al., 1981 ▶; Colby et al., 1995 ▶). Several isoforms of SmDex, as well as other dextranases belonging to GH 66, have been detected (Igarashi et al., 1995 ▶; Khalikova et al., 2005 ▶) and our recent work has also shown that recombinant full-length SmDex (Ser25–Asp850; 95 kDa) expressed in Escherichia coli is susceptible to protease digestion after long storage (Kim et al., 2011 ▶). Since the production of stable enzymes is a prerequisite for further biochemical studies and industrial applications, we have constructed a truncation mutant tolerant to protease digestion which is devoid of the N- and C-­terminal variable regions (SmDexTM; Gln100–Ile732).

In biochemical studies of SmDex and two other GH 66 proteins, Asp385 of SmDex (Igarashi et al., 2002 ▶), Asp270 of CITase from Bacillus circulans T3040 (Yamamoto et al., 2006 ▶) and Asp243 of endodextranase from Thermotoga lettingae TMO (Kim & Kim, 2010 ▶) have been implicated as catalytic residues. However, the detailed catalytic mechanism of GH 66 enzymes is as yet unclear owing to the lack of three-dimensional structures. We recently reported that ω-­epoxyalkyl α-glucopyranosides inactivate SmDex irreversibly, but the mechanism of this suicide inhibitor needs to be further elucidated (Kang et al., 2010 ▶). In order to address these questions and to obtain a structural basis for protein engineering applicable for use in iso­maltooligosaccharide production, we attempted the crystallization of SmDexTM.

2. Materials and methods

SmDexTM was overexpressed with the pET-28 expression system (Novagen, Madison, Wisconsin, USA) using E. coli BL21 (DE3) cells in LB medium by induction with 0.1 mM isopropyl β-d-1-thio­galactopyranoside overnight at 298 K as reported previously (Kim et al., 2011 ▶). The protein was purified from the soluble fraction of the bacterial cell lysate by Ni²⁺-charged HiTrap chelating HP column chromatography (GE Healthcare, Buckinghamshire, England) in a buffer consisting of 50 mM sodium phosphate pH 6.8, 300 mM NaCl, 200 mM imidazole. After desalting the solution using PD-10 column chromatography (GE Healthcare) into a buffer consisting of 20 mM Tris–HCl pH 8.0, the protein was further purified by anion-exchange chromatography using HiTrap Q-Sepharose HP (GE Healthcare) using a buffer consisting of 1 M NaCl, 20 mM Tris–HCl pH 8.0.

The protein solution was desalted and concentrated to 9.8 mg ml⁻¹ by ultrafiltration using a YM-30 membrane (Millipore, Billerica, Massachusetts, USA) and filtered through a 0.1 µm membrane (Millipore). The pooled solution without buffer reagents was used in crystallization trials. Sparse-matrix crystal screening was performed using Crystal Screen HT, Index HT (Hampton Research, Aliso Viejo, California, USA), Wizard I and II and Cryo I and II (Emerald Bio­Systems, Bainbridge Island, Washington, USA). Sitting-drop vapour-diffusion trials were set up using the Thermo Scientific Matrix Hydra II (Thermo Fisher Scientific Inc., Hudson, New Hampshire, USA) automated liquid-handling system in 96-well Intelli-Plates (Art Robbins Instruments, Sunnyvale, California, USA) at 293 K using 50 µl reservoir solution; each drop consisted of 0.3 µl protein solution and 0.3 µl reservoir solution. Within two weeks, several tiny plate-shaped crystals were observed under several conditions using polyethylene glycol 600 or polyethylene glycol monomethyl ether 2000 as a precipitant. Crystallization conditions were optimized manually by refinement of the protein and/or precipitant concentrations and buffer solution using CrystalClear Strips 96-well sitting-drop plates (Douglas Instruments, Berkshire, England).

Native diffraction data were collected from a single crystal on beamline BL-17A of the Photon Factory (PF), High Energy Accelerator Research Organization, Tsukuba, Japan. The crystal was scooped in a nylon CryoLoop (Hampton Research) and then flash-cooled in a nitrogen-gas stream at 95 K. Diffraction data were collected with 5 s exposures for 1° oscillations over a total of 360° at a wavelength of 0.970 Å with a Quantum 270 CCD detector (ADSC, Poway, California, USA). Data were integrated and scaled using the programs DENZO and SCALEPACK from the HKL-2000 program suite (Otwinowski & Minor, 1997 ▶).

3. Results and discussion

We optimized the crystallization condition for SmDexTM, which was composed of 30%(w/v) polyethylene glycol monomethyl ether 2000, 100 mM phosphate–citrate buffer pH 4.2. The plate-shaped crystals were obtained in a drop consisting of 2 µl protein solution with a protein concentration of 3.6 mg ml⁻¹ and 2 µl reservoir solution equilibrated against 50 µl reservoir solution at 293 K. The largest crystal grew to dimensions of 300 × 150 × 30 µm within one month (Fig. 1 ▶).

Figure 1.

The crystal of SmDexTM. The scale bar represents 100 µm.

The crystal diffracted to a maximum resolution of 1.6 Å using synchrotron radiation at PF. The crystal belonged to space group P2₁, with unit-cell parameters a = 53.2, b = 89.7, c = 63.3 Å, β = 102.3°. The processing statistics of the collected data are summarized in Table 1 ▶. Assuming that the asymmetric unit of the crystal contained one SmDexTM molecule, the Matthews coefficient was calculated to be 4.07 ų Da⁻¹ (Matthews, 1968 ▶); assuming two molecules to be present in the asymmetric unit it was calculated to be 2.03 ų Da⁻¹.

Table 1. Data-collection statistics for the SmDexTM crystal.

Values in parentheses are for the highest resolution shell.

Beamline	PF BL-17
Detector	ADSC Quantum 270
Wavelength (Å)	0.970
Space group	P21
Unit-cell parameters (Å, °)	a = 53.2, b = 89.7, c = 63.3, β = 102.3
Resolution range (Å)	50.0–1.6 (1.66–1.60)
Rmerge†	0.063 (0.175)
Completeness (%)	100.0 (100.0)
Multiplicity	7.5 (7.5)
Average I/σ(I)	39.2 (11.5)
Unique reflections	76683 (7641)
Total reflections	571898
Mosaicity (°)	0.77
Overall B factor from Wilson plot (Å2)	13.5

^† R _merge = , where I_i(hkl) is the intensity of the ith observation of reflection hkl and 〈I(hkl)〉 is the weighted average intensity for all i observations of reflection hkl.

Although three-dimensional structures of GH 66 enzymes have not been reported, several crystal structures of enzymes belonging to GH 13, GH 27 and GH 31, which share a common catalytic (β/α)₈-barrel, are available. We performed molecular-replacement calculations with the program MOLREP (Vagin & Teplyakov, 2010 ▶; Winn et al., 2011 ▶) using polyalanine structures of the conserved regions, including those of two GH 13 α-amylases from B. subtilis (PDB entry 1bag; Fujimoto et al., 1998 ▶) and from Geobacillus stearothermophilus (PDB entry 1hvx; Suvd et al., 2001 ▶), two GH 27 α-galactosidases from rice (PDB entry 1uas; Fujimoto et al., 2003 ▶) and from Umbelopsis vinacea (PDB entry 3a5v; Fujimoto et al., 2009 ▶) and GH 31 α-xylosidase (PDB entry 1we5; Kitamura et al., 2005 ▶). All of the trials gave a single solution, indicating that the crystal contains one SmDexTM molecule in the asymmetric unit, with a Matthews coefficient of 4.07 ų Da⁻¹ corresponding to 69.8% solvent content. However, the solutions were insufficient to solve the structure because of their low amino-acid sequence identities and the lack in these structures of the extra domains possessed by SmDexTM. We are currently preparing a selenomethionine-substituted protein crystal (17 methionines per molecule) for phase determination using the multiple anomalous dispersion technique.