Expression, crystallization and preliminary X-ray crystallographic analysis of alcohol dehydrogenase (ADH) from Kangiella koreensis

Alcohol dehydrogenase (ADH) catalyzes the oxidation of alcohols using NAD(P)⁺ as a cofactor. The adh gene from K. koreensis was cloned and the protein was expressed, purified and crystallized. A preliminary X-ray crystallographic analysis of the ADH crystal was performed.

Abstract

Alcohol dehydrogenases (ADHs) are a group of dehydrogenase enzymes that facilitate the interconversion between alcohols and aldehydes or ketones with the reduction of NAD⁺ to NADH. In bacteria, some alcohol dehydrogenases catalyze the opposite reaction as part of fermentation to ensure a constant supply of NAD⁺. The adh gene from Kangiella koreensis was cloned and the protein (KkADH) was expressed, purified and crystallized. A KkADH crystal diffracted to 2.5 Å resolution and belonged to the monoclinic space group P2₁, with unit-cell parameters a = 94.1, b = 80.9, c = 115.6 Å, β = 111.9°. Four monomers were present in the asymmetric unit, with a corresponding V _M of 2.55 ų Da⁻¹ and a solvent content of 51.8%.

1. Introduction  

Alcohol dehydrogenases (ADHs; EC 1.1.1.1) are a group of dehydrogenase enzymes that occur in many organisms and facilitate the interconversion between alcohols and aldehydes or ketones with the reduction of NAD⁺ to NADH (Nosova et al., 1997 ▶). In bacteria, some alcohol dehydrogenases catalyze the opposite reaction as part of fermentation to ensure a constant supply of NAD⁺ (Wales & Fewson, 1994 ▶). ADHs exist as dimers that comprise 40 kDa subunits and contain zinc in their catalytic site.

ADHs have been widely investigated in various organisms. Unlike alcohol dehydrogenases from eukaryotes, only a few structures of prokaryotic ADHs have been identified to date. Among the reported ADHs, an ADH from a frog (ADH8) is the only alcohol dehydro­genase that has specificity towards NADP(H) instead of NAD(H) (Peralba et al., 1999 ▶). A BLASTP search at NCBI using ADH8 as a template was performed to search for a bacterial homologue of ADH8 that may share similar activity with ADH8. The BLASTP results showed that ADH from Kangiella koreensis shared the highest sequence identity (48.9%) with ADH8.

K. koreensis is a Gram-negative, nonmotile, non-spore-forming bacterium isolated from tidal flat sediments at Daepo Beach, Yellow Sea, Korea (Han et al., 2009 ▶). Its phylogeny is of interest because of the very isolated location of the genus Kangiella in the gammaproteobacterial order Oceanospirillales (Han et al., 2009 ▶). In order to obtain a better understanding of the specific function of K. koreensis ADH, the adh gene was cloned into Escherichia coli ER2566 using the pRSFDuet-1 vector. This study describes the expression, purification, crystallization and preliminary X-ray crystallographic studies of K. koreensis ADH.

2. Materials and methods  

2.1. Cloning  

The genomic DNA from K. koreensis DSM 16069 was extracted using a genomic DNA extraction kit (Qiagen, Hilden, Germany). The Kkadh gene (1123 bp) encoding the alcohol dehydrogenase was amplified by PCR using the genomic DNA isolated from K. koreensis as a template. The sequences of the oligonucleotide primers used for gene cloning were based on the DNA sequence of K. koreensis alcohol dehydrogenase (GenBank accession No. NC_013166). Forward (5′-GGATCCAATGTCGAACGAAGTGATTAAATG-3′) and reverse (5′-CTCGAGATAATGAATCACGCTACGAATA-3′) primers were designed to introduce BamHI and XhoI restriction sites (bold), respectively. The PCR-amplified DNA fragments were purified using a QIAquick gel-extraction kit (Qiagen, Hilden, Germany), inserted into the pRSFDuet-1 vector digested with the same restriction enzymes and ligated into the BamHI and XhoI sites of pRSFDuet-1, yielding the recombinant clone pRSFDuet-1-KkADH. The pRSFDuet-1-KkADH plasmid was transformed into E. coli ER2566 and plated on Luria–Bertani (LB) agar containing 20 µg ml⁻¹ kanamycin. A kanamycin-resistant colony was selected and plasmid DNA from the transformant was isolated using a plasmid purification kit (Promega, Madison, Wisconsin, USA). DNA sequencing was carried out at the Macrogen facility (Seoul, Republic of Korea).

2.2. Overexpression and purification  

The recombinant E. coli cells containing pRSFDuet-1-KkADH coding for residues 1–374 were cultivated in a 2 l flask containing 500 ml LB medium and 20 µg ml⁻¹ kanamycin at 310 K with shaking at 200 rev min⁻¹. When the optical density of the bacteria reached 0.6 at 600 nm, isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM to induce expression of KkADH. The culture was incubated with shaking at 150 rev min⁻¹ and 288 K for an additional 12 h to obtain the highest expression level. The cells were harvested by centrifugation for 20 min at 6000g and 277 K, washed twice with 0.85% NaCl and then resuspended in 50 mM phosphate buffer containing 300 mM KCl and 10 mM imidazole. The resuspended cells were disrupted on ice using a sonicator (Sonic Vibra Cell, Sonics & Materials Inc.). The unbroken cells and cell debris were removed by centrifugation at 13 000g for 10 min at 277 K and the supernatant was filtered through a 0.45 µm filter.

The filtrate was applied onto an immobilized metal-ion affinity chromatography cartridge (Bio-Rad, Hercules, California, USA) equilibrated with 50 mM phosphate buffer pH 8.0. The cartridge was washed extensively with the same buffer and the bound protein was eluted with a linear gradient from 10 to 250 mM imidazole at a flow rate of 1 ml min⁻¹. The eluate was collected and immediately loaded onto a Bio-Gel P-6 desalting cartridge (Bio-Rad) equilibrated with 50 mM piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) buffer pH 7.5. The loaded protein was eluted with 50 mM PIPES buffer pH 7.5 at flow rate of 1 ml min⁻¹ and the active fractions were collected. The resulting solution was used as the purified enzyme. The homogeneity of the purified protein was analyzed via SDS–PAGE (Fig. 1 ▶). 13 additional residues (GSSHHHHHHSQDP) from the pRSF Duet-­1 vector remained at the N-terminus of KkADH. For crystallization, purified KkADH was dialyzed for 4 h in buffer A (25 mM Tris pH 7.5, 15 mM NaCl, 3 mM β-mercaptoethanol) and concentrated to a final concentration of 8 mg ml⁻¹.

Figure 1.

Purified K. koreensis ADH is shown on a 12% SDS–PAGE gel. Lane M, molecular-mass markers (labelled in kDa); lane P, KkADH.

2.3. Crystallization and X-ray data collection  

Initial crystallization was carried out at 287 K by the sitting-drop vapour-diffusion method in 96-well Intelli-Plates (Art Robbins) using a Hydra II eDrop automated pipetting system (Matrix) and screening kits from Hampton Research (Index, Crystal Screen, Crystal Screen Cryo, Crystal Screen Lite, PEGRx 1 and PEGRx 2), Emerald BioSystems (Wizard Classic 1 and 2) and Molecular Dimensions (Morpheus). 0.5 µl protein solution was mixed with 0.5 µl reservoir solution and equilibrated against 70 µl reservoir solution. After 3 d, multiple thin needle-shaped crystals (with the appearance of broom heads) were observed in condition F12 of the Index kit from Hampton Research [0.1 M sodium chloride, 0.1 M HEPES pH 7.5, 25%(w/v) PEG 3350; Fig. 2 ▶ a]. Crystals were reproduced by the sitting-drop method, in which drops consisting of 0.9 µl protein solution mixed with 0.9 µl reservoir solution were equilibrated against 1 ml reservoir solution. Optimization was achieved by varying the concentration of PEG 3350, the pH and the incubation temperature. Crystals with adequate dimensions were obtained after 5 d at 303 K using a reservoir solution consisting of 0.1 M sodium chloride, 0.1 M HEPES pH 8.0, 24%(w/v) PEG 3350 (Fig. 2 ▶ b). The fully grown crystals (0.1 × 0.07 × 0.008 mm) were flash-cooled at 100 K in liquid nitrogen using 20%(v/v) glycerol, 0.1 M sodium chloride, 0.1 M HEPES pH 8.0, 24%(w/v) PEG 3350 as a cryoprotectant. X-ray diffraction data were collected from the cryoprotected crystal (at 100 K) on an ADSC Q315r detector using 1° oscillations with a crystal-to-detector distance of 400 mm on beamline 5C SBII at the Pohang Light Source (PLS), Republic of Korea. The crystals diffracted to 2.5 Å resolution. A diffraction image is shown in Fig. 3 ▶. Diffraction data were integrated and scaled using the HKL-2000 program suite (Otwinowski & Minor, 1997 ▶).

Figure 2.

Crystals of K. koreensis ADH. (a) Initial crystals obtained after 3 d using a reservoir solution consisting of 0.1 M sodium chloride, 0.1 M HEPES pH 7.5, 25%(w/v) PEG 3350 from the Index kit (Hampton Research). (b) Optimized crystal of KkADH with dimensions of 0.1 × 0.07 × 0.008 mm obtained using a condition consisting of 0.1 M sodium chloride, 0.1 M HEPES pH 8.0, 24%(w/v) PEG 3350. The scale bar represents 0.02 mm.

Figure 3.

X-ray diffraction image from a crystal of K. koreensis ADH.

3. Results and discussion  

Like other ADHs, KkADH eluted in a dimeric form in size-exclusion chromatography. Initial crystals obtained from sparse-matrix crystallization screening were multiple thin needles with the appearance of broom heads. Crystals with adequate dimensions were obtained after one month by the sitting-drop method at room temperature (Fig. 2 ▶ b). A shorter growth time was achieved (5 d) by changing the incubation temperature from room temperature to 303 K.

The crystal belonged to the monoclinic space group P2₁. The unit-cell parameters were a = 94.1, b = 80.9, c = 115.6 Å, β = 111.9°. The space group was assigned by auto-indexing (Otwinowski & Minor, 1997 ▶) and data-collection statistics are provided in Table 1 ▶. According to calculation of the Matthews coefficient (Matthews, 1968 ▶), there are probably four molecules in the asymmetric unit, with a V _M of 2.55 ų Da⁻¹ and a solvent content of 51.8%. Self-rotation functions were calculated at χ = 180, 120, 90 and 60° to detect twofold, threefold, fourfold and sixfold symmetry, respectively. The self-rotation function was calculated using data from 50 to 4 Å resolution in MOLREP (Vagin & Teplyakov, 2010 ▶). A strong peak was displayed in the χ = 180° section, indicating the presence of twofold noncrystallographic symmetry (Fig. 4 ▶). The presence of pseudo-translation was also detected by MOLREP. Molecular replacement (MR) using Phaser in the CCP4 program package (McCoy et al., 2007 ▶) with human glutathione-dependent formaldehyde dehydro­genase (PDB entry 2fze; 69.3% sequence identity; Lang et al., 2012 ▶) as a search model was successful and showed that four monomers were present in the asymmetric unit. The initial R value from the MR solution was 49.2%. The resulting electron-density maps were clear and fitted the main chain of the model well. No clashes were found between molecules. Rigid-body and restrained refinement were performed in REFMAC5 (Murshudov et al., 2011 ▶), which decreased the R value to 35.7% and R _free to 42.7%. The side chains that were outside the electron-density map were re-fitted and the coordinates were again input to REFMAC5 to run restrained refinement. After several cycles of fitting and refining, the R value decreased to 19.5% and R _free to 25.5%. Currently, the structure is being refined. Final structural details will be described in a separate paper.

Table 1. Data-collection statistics.

Values in parentheses are for the outer shell.

X-ray source	Beamline 5C SBII, PLS
Wavelength (Å)	0.97951
Unit-cell parameters (Å, °)	a = 94.1, b = 80.9, c = 115.6, α = γ = 90.0, β = 111.9
Total rotation (°)	360
Mosaicity (°)	0.6
Multiplicity	7.2 (5.3)
Space group	P21
Resolution	50.0–2.5 (2.54–2.50)
Total No. of observations	400525
No. of unique observations	55363
Completeness (%)	99.3 (90.6)
R merge † (%)	7.4 (38.8)
〈I/σ(I)〉	42.8 (5.3)

^† R _merge = , where I_i(hkl) is the intensity of the ith observation of reflection hkl, is the sum over all reflections and is the sum over i measurements of reflection hkl.

Figure 4.

Self-rotation function at χ = 180° calculated for the KkADH crystal.