Purification, crystallization and preliminary X-ray analysis of two hydrogen sulfide-producing enzymes from Fusobacterium nucleatum

Two homologous hydrogen sulfide-producing enzymes, Fn1220 and Cdl, from F. nucleatum (which actively produces hydrogen sulfide) were overproduced, purified and crystallized. The crystals obtained were characterized by X-ray diffraction.

Abstract

Hydrogen sulfide produced by oral bacteria is responsible for oral malodour. Two homologous hydrogen sulfide-producing enzymes, Fn1220 and Cdl, from Fusobacterium nucleatum (which actively produces hydrogen sulfide) were overproduced, purified and crystallized. X-ray diffraction data were collected from the crystals using a synchrotron-radiation source. The Fn1220 crystal belonged to tetragonal space group P4₁2₁2 or P4₃2₁2 (unit-cell parameters a = b = 116.8, c = 99.2 Å) and the Cdl crystal belonged to monoclinic space group P2₁ (unit-cell parameters a = 84.9, b = 70.9, c = 87.6 Å, β = 90.3°).

1. Introduction  

Hydrogen sulfide (H₂S), a pungent gas, is one of the predominant volatile sulfur compounds responsible for oral malodour. This gas is produced by the enzymatic actions of oral bacteria. It is known that periodontal pathogens generally produce significant amounts of H₂S. Among them, Fusobacterium nucleatum is one of the most active H₂S producers (Persson et al., 1990 ▶; Yoshida et al., 2009 ▶) and is the most commonly occurring species in the periodontal gingival pocket (Moore & Moore, 1994 ▶). Therefore, the H₂S produced by F. nucleatum may be associated with the aetiology of periodontitis.

A recently developed novel detection method for enzymes that produce H₂S based on SDS–polyacrylamide gel electrophoresis (Yoshida, Ito, Tamura et al., 2010 ▶) has enabled us to confirm the presence of multiple H₂S-producing enzymes in F. nucleatum species (Yoshida, Ito, Kamo et al., 2010 ▶) with greater clarity. Currently, four genes (fn0625, fn1055, fn1220 and fn1419) encoding pyridoxal-5′-­phosphate (PLP) dependent H₂S-producing enzymes have been identified in F. nucleatum subsp. nucleatum ATCC 25586 (Yoshida, Ito, Kamo et al., 2010 ▶; Yoshida et al., 2011 ▶; Suwabe et al., 2011 ▶) and one gene (cdl) has been identified in subsp. polymorphum ATCC 10953 (Fukamachi et al., 2002 ▶). fn1220 is a cdl orthologue, and the gene products Fn1220 and Cdl share 92% identity at the amino-acid level. Fn1220 catalyses the β-replacement reaction (Fig. 1 ▶) which condenses two l-cysteine molecules into the uncommon amino acid l-­lanthionine to produce H₂S (Yoshida, Ito, Kamo et al., 2010 ▶). Of the four enzymes identified in F. nucleatum ATCC 25586, Fn1220 displayed not only the highest turnover rate but also the highest transcription level of the gene. The amount of H₂S produced by Fn1220 was estimated to be 87.6% of the total H₂S produced from l-­cysteine (Suwabe et al., 2011 ▶).

Figure 1.

Putative reaction mechanism for production of H₂S and l-lanthionine by Fn1220 and Cdl. Lys42 is a probable PLP-binding residue (internal aldimine). Subsequent chemical transformations of PLP by Fn1220 and Cdl were considered by analogy with those performed by an O-acetyl-l-serine sulfhydrylase (Schnell et al., 2007 ▶), which also catalyses a β-­replacement reaction of an amino acid.

The potentially significant contribution of Fn1220 to H₂S production from l-cysteine suggests that a deeper understanding of the reaction mechanism of Fn1220 and Cdl will provide important insights into H₂S production in F. nucleatum. Here, we describe the purification, crystallization and preliminary X-ray crystallographic analysis of Fn1220 and Cdl from F. nucleatum species.

2. Materials and methods  

2.1. Sample preparation and crystallization  

Recombinant Fn1220 was overproduced in Escherichia coli as a glutathione S-transferase (GST) fused protein and was purified using glutathione Sepharose 4B (GE Healthcare), as reported previously (Yoshida, Ito, Kamo et al., 2010 ▶). For further purification, protein without GST tag was loaded onto Mono Q HR 10/10 (GE Healthcare) equilibrated with 20 mM Tris–HCl pH 8.5 containing 150 mM NaCl. The bound protein was eluted using a linear gradient of NaCl (150–350 mM over eight column volumes) at a flow rate of 2.0 ml min⁻¹ and 1.5 ml fractions were collected. The Fn1220-containing fractions were concentrated and applied onto HiLoad 16/60 Superdex 200 pg (GE Healthcare). Phosphate-buffered saline (PBS) was used as the mobile phase and was pumped at a flow rate of 1.0 ml min⁻¹. The purified Fn1220 was dialysed against 10 mM Tris–HCl pH 7.6 containing 10 mM NaCl and 10 µM PLP, and was then concentrated and adjusted to 10.0 mg ml⁻¹ on the basis of the extinction coefficient at 280 nm (Pace et al., 1995 ▶).

cdl (Fukamachi et al., 2002 ▶) corresponding to residues 4–301 was amplified from genomic DNA of F. nucleatum subsp. polymorphum ATCC 10953 using the forward primer 5′-GGGGATCCAATTCT­GTAATTGATTTAATTGGGAACACC-3′ and the reverse primer (5′-GGCTCGAGTTAAGATAGATATTTTTCTCCTGAG­TCAGT-3′). The product was then ligated into pGEX-6P-2 (GE Healthcare) at the BamHI and XhoI sites, juxtaposing cdl downstream of the coding sequence for GST and the PreScission protease recognition site. Overproduction and purification of Cdl was performed using the same procedure as used for Fn1220 (Yoshida, Ito, Kamo et al., 2010 ▶), except for the use of CHT Ceramic Hydroxyapatite (Bio-Rad) packed in a Tricorn 10/100 column (GE Healthcare) instead of Mono Q HR 10/10. After purification using glutathione Sepharose 4B and removal of the GST tag using PreScission protease, Cdl was loaded onto a 7 ml hydroxyapatite column equilibrated with 5 mM potassium phosphate buffer pH 7.0. The bound protein was eluted using a linear gradient of potassium phosphate (5–400 mM over 20 column volumes) at a flow rate of 5.0 ml min⁻¹.

Initial screening for crystallization conditions was performed using the hanging-drop vapour-diffusion method at 293 K. Aliquots of 0.8 µl protein solution and 0.8 µl reservoir solution were equilibrated against 200 µl reservoir solution in a 48-well plate. Screening was conducted using commercially available kits produced by Emerald BioSystems and Hampton Research. The initial crystallization conditions were further optimized by changing the concentrations of the precipitant, salt and/or buffer.

The purified enzymes (0.4 mg) were analysed by gel-filtration chromatography on Superdex 200 HR 10/30 (GE Healthcare) at a flow rate of 0.25 ml min^–1 in PBS. The molecular weights were estimated using a standard curve based on molecular-weight markers (Gel Filtration Markers Kit for Protein Molecular Weights 12 000–200 000 Da, Sigma–Aldrich). Elution of the enzyme was monitored at 280 nm.

2.2. Data collection and processing  

The crystals were mounted in nylon loops and flash-cooled in a stream of nitrogen gas at 90 K. X-ray diffraction data for Fn1220 and Cdl were collected on beamline BL5A of the Photon Factory, Ibaraki, Japan using a Quantum 210r CCD detector (Area Detector Systems Corporation). The diffraction data were indexed, integrated and scaled using MOSFLM (Powell, 1999 ▶) and SCALA (Evans, 2006 ▶) as implemented in xia2 (Winter, 2010 ▶). Subsequent data analysis was performed using the CCP4 suite (Winn et al., 2011 ▶).

2.3. Self-rotation function  

The self-rotation functions were calculated by MOLREP (Vagin & Teplyakov, 2010 ▶) with a maximum resolution of 3.0 Å. The integration radii were set to 25.0 Å.

3. Results and discussion  

Recombinant Fn1220 and Cdl were expressed in E. coli and purified to apparent homogeneity. The gel-filtration analysis showed single peaks for purified Fn1220 and Cdl, with retention volumes corresponding to 64.1 and 56.5 kDa, respectively (Fig. 2 ▶). These values indicated that the enzymes, which have theoretical molecular weights of 33 kDa, are present as homodimers in solution.

Figure 2.

Gel-filtration chromatography of Fn1220 (a) and Cdl (b) on Superdex 200 HR 10/30. Major peaks at a retention volume of approximately 12–13 ml correspond to each protein. The retention volumes of the molecular-weight standards (from the left: 200, 150, 66, 29 and 12 kDa) are shown above each chromatogram.

After initial screening trials using approximately 900 conditions, we obtained tetragonal bipyramidal crystals of Fn1220 from condition No. 6 of the Cryo I kit [40%(v/v) polyethylene glycol with an average molecular weight of 600 (PEG 600), 100 mM cacodylate pH 6.5, 0.2 M calcium acetate]. The optimized composition of the reservoir solution was 41.0%(v/v) PEG 600, 0.1 M HEPES pH 7.5, 0.17 M calcium acetate monohydrate. Crystals grew within 3 d under these conditions, reaching maximum dimensions of 100 × 100 × 100 µm (Fig. 3 ▶ a). Because of the high concentration of PEG 600 in the mother liquor, selected crystals in nylon loops could be directly flash-cooled in a stream of nitrogen gas. A complete data set from a crystal was collected to 2.03 Å resolution. The Laue group was found to be 4/mmm and the unit-cell parameters were a = b = 116.8, c = 99.2 Å. Only reflections with h = 2n, k = 2n and l = 4n were observed along the (h00), (0k0) and (00l) axes, respectively, indicating the tetragonal space group to be P4₁2₁2 or P4₃2₁2. The calculated Matthews coefficient V _M (Matthews, 1968 ▶) and solvent content were 2.5 ų Da⁻¹ and 51%, respectively. This result clearly suggests the presence of two Fn1220 monomers (one dimer) in the asymmetric unit, which is consistent with the gel-filtration analysis. Table 1 ▶ summarizes the processing statistics of the collected data.

Figure 3.

Crystals of Fn1220 (a) and Cdl (b). The scale bars correspond to 200 µm.

Table 1. Data-collection and processing statistics.

Values in parentheses are for the highest resolution shell.

Protein	Fn1220	Cdl
Experimental conditions
Beamline	BL5A	BL5A
Wavelength (Å)	1.0000	1.0000
Temperature (K)	95	95
Detector	ADSC Quantum 210r	ADSC Quantum 210r
Oscillation angle (° per frame)	1.0	1.0
No. of images	360	360
Crystal parameters
Space group	P41212 or P43212	P21
Unit-cell parameters (Å, °)	a = b = 116.82, c = 99.19	a = 84.93, b = 70.86, c = 87.61, β = 90.3
Solvent content (%)	51	39
Monomers per asymmetric unit	2	4
Data processing
Software	MOSFLM/SCALA	MOSFLM/SCALA
Resolution range (Å)	63.50–2.03 (2.08–2.03)	46.29–1.92 (1.97–1.92)
R merge † (%)	6.1 (23.9)	7.5 (22.0)
Completeness (%)	99.9 (100.0)	99.6 (100.0)
〈I/σ(I)〉	9.6 (3.2)	6.2 (3.1)
No. of observed reflections	1179044 (82151)	569619 (41881)
No. of unique reflections	44852 (3255)	79253 (5875)
Multiplicity	26.3 (25.2)	7.2 (7.1)

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

Cdl was crystallized using condition No. 38 of Ozma 4K [200 mM sodium fluoride, 20%(w/v) PEG 4000]. The optimized crystallization solution was found to consist of 0.25 M sodium fluoride, 24%(w/v) PEG 3350. Under this condition, rod-like crystals with dimensions of 200 × 50 × 50 µm grew within several days (Fig. 3 ▶ b). The most highly ordered crystal diffracted to 1.92 Å resolution. The Laue group was 2/m and the unit-cell parameters were a = 84.9, b = 70.9, c = 87.6 Å, β = 90.3°. Only reflections with k = 2n were observed along the (0k0) axis, indicating the monoclinic space group P2₁. Three or four monomers were expected to be present in the asymmetric unit based on a V _M of 2.7 or 2.0 ų Da^–1, respectively. As suggested by the gel-filtration chromatography results (Fig. 2 ▶ b), Cdl was estimated to be a dimeric protein. Therefore, it is most likely that four monomers (two dimers) are present in the asymmetric unit. The processing statistics of the collected data are presented in Table 1 ▶. The self-rotation map showed three crystallographic independent peaks, representing noncrystallographic twofold axes, on the χ = 180° section (Fig. 4 ▶). We confirmed that axis 2 [(θ, ϕ) = (26.7°, 0.0°)] was related to axis 3 (25.3°, −106.2°) by the twofold-symmetry operation of axis 1 (91.0°, 39.3°). Therefore, axes 2 and 3 were considered to be located between subunits in each dimer, and the two dimers were then related by the third noncrystallographic twofold axis (axis 1).

Figure 4.

Stereographic projections along the c axis of the χ = 180° polar section of the self-rotation function for Cdl. Latitude (θ angle) and longitude (ϕ angle) grid lines are drawn at 10° intervals. The independent noncrystallographic twofold axis peaks are indicated by numbers (1–3). The labels 1′, 2′ and 3′ correspond to the symmetrically equivalent noncrystallographic twofold axis peaks.

The crystals of Fn1220 and Cdl were of sufficiently high quality for structure determination at high resolution. Fn1220 and Cdl share a sequence identity of 46% with O-acetyl-l-serine sulfhydrylase from Mycobacterium tuberculosis (PDB entry 2q3b; Schnell et al., 2007 ▶). Therefore, this structure was considered to be a suitable template for the construction of search models. Phase determination performed by the molecular-replacement technique is currently in progress.