Crystallization and preliminary X-ray analysis of formate oxidase, an enzyme of the glucose–methanol–choline oxidoreductase family

Formate oxidase from A. oryzae RIB40 was crystallized and diffraction data were collected to a resolution of 2.4 Å.

Abstract

Formate oxidase (FOD), which catalyzes the oxidation of formate to yield carbon dioxide and hydrogen peroxide, belongs to the glucose–methanol–choline oxidoreductase (GMCO) family. FOD from Aspergillus oryzae RIB40, which has a modified FAD as a cofactor, was crystallized at 293 K by the hanging-drop vapour-diffusion method. The crystal was orthorhombic and belonged to space group C222₁. Diffraction data were collected from a single crystal to 2.4 Å resolution.

1. Introduction

The enzymes of the glucose–methanol–choline oxidoreductase (GMCO) family exhibit little sequence similarity in their catalytic sites. However, either a His–Asn or a His–His pair is conserved in the catalytic site of all enzymes belonging to the GMCO family. The crystal structures of choline oxidase from Arthrobactor globiformis (Quaye et al., 2008 ▶), glucose oxidase from Aspergillus niger (Hecht et al., 1993 ▶), cholesterol oxidase from Brevibacterium stero­licum (Vrielink et al., 1991 ▶; Lario et al., 2003 ▶), the flavin domain of cellobiose dehydrogenase from Phanerochaete chrysosporium (Hallberg et al., 2002 ▶) and pyranose 2-oxidase from Peniophora sp. (Bannwarth et al., 2004 ▶) show that they all share a highly conserved catalytic site.

Enzymes that are capable of oxidizing formate to carbon dioxide are classified as formate dehydrogenases and formate oxidases (FODs). Formate dehydrogenases from various microorganisms and higher plants have been well characterized (Boyington et al., 1997 ▶; Moura et al., 2004 ▶; Tishkov & Popov, 2006 ▶; Schirwitz et al., 2007 ▶; Shabalin et al., 2009 ▶). In contrast, there are only a few reports on FODs. FOD from Aspergillus nomius IRI013 was the first FOD to be purified and characterized (Kondo et al., 2002 ▶). Recently, we purified and characterized FOD from Debaryomyces vanrijiae MH201 and subsequently cloned and expressed three FOD genes from D. vanrijiae MH201 in Escherichia coli (Uchida et al., 2007 ▶; Maeda et al., 2008 ▶). An FOD gene from A. oryzae RIB40 was also expressed in E. coli (Maeda et al., 2009 ▶). The native and recombinant enzymes are all dimeric proteins with subunit molecular masses of about 64 kDa. In the amino-acid sequences deduced from their nucleotide sequences, three FAD-binding site motifs and a His residue are conserved throughout the GMCO family (Dym & Eisenberg, 2001 ▶; Fan et al., 2004 ▶; Ghanem & Gadda, 2005 ▶).

The amino-acid sequence deduced from the nucleotide sequence of the FOD gene from A. oryzae RIB40 shows 28.9 and 25.7% similarity to those of choline oxidase from Arthrobactor globiformis and glucose oxidase from Aspergillus niger, respectively (Fig. 1 ▶). A His511–Arg554 pair might exist in the catalytic site of the recombinant A. oryzae FOD (FOD_AO), instead of the His–Asn and His–His pairs that exist in the catalytic sites of the choline and glucose oxidases, respectively. Also, His508 and Arg551, which correspond to His511 and Arg554, respectively, are found in FOD from D. vanrijiae MH201. The choline and glucose oxidases contain FAD that is covalently bound to His97 via the 8α-position of the isoalloxazine ring and noncovalently bound, respectively (Hecht et al., 1993 ▶; Quaye et al., 2008 ▶). Spectroscopic analyses of FOD_AO and an extract that was obtained by boiling the enzyme indicated that FOD_AO has a modified noncovalently bound FAD with a molecular mass of 799, which was expected to be 8-formyl-FAD (Maeda et al., 2009 ▶). The spectroscopic properties of the extract derived from wild-type FOD purified from D. vanrijiae MH201 were identical to those of FOD_AO. The modification of FAD as well as the presence of the His–Arg pair in the catalytic site might be characteristic of FOD. In this report, we describe the crystallization and preliminary X-ray diffraction studies of FOD_AO in order to obtain insight into the structures of its catalytic site and modified FAD.

Figure 1.

Sequence alignment of FOD_AO with choline oxidase from Arthrobactor globiformis (NCBI accession No. AAS99880) and glucose oxidase from Aspergillus niger (AAF59929). The alignment was constructed using ClustalW2. Gaps were introduced into the sequences to maximize the homology and are indicated by dashes. Identical residues are labelled with asterisks (*). Homologous and semi-homologous residues are labelled with ‘:’ and ‘.’, respectively. The three FAD-binding motifs are indicated by boxes. The two active-site residues are indicated in bold.

2. Experimental procedure

2.1. Protein expression and purification

E. coli BL21 (DE3) cells harbouring plasmid pET-FOD _AO were grown and induced with 0.1 mM isopropyl β-d-1-thiogalactopyranoside as described previously (Maeda et al., 2009 ▶). The cells were harvested by centrifugation, suspended in 10 mM acetate buffer pH 6.0 and disrupted using an ultrasonic generator. Cell debris was removed by centrifugation. The supernatant was applied onto a column of Ni Sepharose 6 Fast Flow (bed volume 10 ml; GE Healthcare, UK) equilibrated with 10 mM acetate buffer pH 6.0. The column was washed with the equilibration buffer and FOD_AO was eluted with 500 mM imidazole buffer pH 6.2. Fractions showing FOD activity were combined and dialyzed against 10 mM phosphate buffer pH 7.0. The dialyzate was applied onto a HiTrap QHP column (5 ml; GE Healthcare, UK) that had been equilibrated with 10 mM phosphate buffer pH 7.0. After washing the column with the equilibration buffer, FOD_AO was eluted with a linear gradient of 0–0.5 M NaCl in 10 mM phosphate buffer pH 7.0. The active fractions were pooled and applied onto a HisTrap FF column (1 ml; GE Healthcare, UK) equilibrated with 10 mM phosphate buffer pH 7.0. The column was washed with the equilibration buffer and FOD_AO was eluted with 500 mM imidazole buffer pH 6.2. The active fractions were collected and dialyzed against 10 mM acetate buffer pH 6.0. The dialyzate was concentrated by ultrafiltration using Ultrafree-MC (Millipore Co., Tokyo, Japan) and used for crystallization.

2.2. Crystallization

Initial screening for FOD_AO crystallization was performed at 293 K by the sitting-drop vapour-diffusion method using Crystal Screen, Crystal Screen 2 and Crystal Screen Lite from Hampton Research (Laguna Niguel, California, USA) with 96-well plates. Low-quality crystals of the enzyme were obtained from condition Nos. 37 [0.1 M sodium acetate trihydrate pH 4.6, 4%(w/v) PEG 4000] and 40 [0.1 M sodium citrate tribasic dyhydrate pH 5.6, 10%(v/v) 2-propanol, 10%(w/v) PEG 4000] from Crystal Screen Lite. Further screening was carried out at 277 and 293 K by modification of the PEG concentration and by replacement of the buffer. After improvement of the conditions, final crystallization was performed by the hanging-drop vapour-diffusion method using 24-well plates at 293 K. The hanging drop contained 2 µl enzyme solution (35 mg ml⁻¹) and 2 µl reservoir solution [0.1 M sodium acetate buffer pH 4.6 and 6%(w/v) PEG 4000] and was equilibrated against 1 ml reservoir solution.

2.3. X-ray data collection

A crystal of FOD_AO was picked up from a droplet and transferred into cryoprotectant solution [0.1 M sodium acetate buffer pH 4.6, 6%(w/v) PEG 4000 and 30%(w/v) 2-methyl-2,4-pentanediol]. The crystal was mounted on a nylon loop (Hampton Research, Laguna Niguel, California, USA) and immediately flash-cooled in a cold nitrogen-gas stream at 100 K. X-ray diffraction images were checked using an in-house Bruker High-Star multi-wire detector and a rotating-anode X-ray generator. The final diffraction data for the selected crystal were collected on a Rigaku R-AXIS V detector using synchrotron radiation of wavelength 1.00 Å at the BL-26B1 station of SPring-8 (Hyogo, Japan; Ueno et al., 2006 ▶). The collected data were processed with HKL-2000 (Otwinowski & Minor, 1997 ▶).

3. Results and discussion

A diamond-shaped crystal of FOD_AO appeared in two weeks (Fig. 2 ▶). The crystal yielded good diffraction images as shown in Fig. 3 ▶. The crystal parameters and diffraction data statistics are summarized in Table 1 ▶. The space group of the crystal was determined to be C222₁, with unit-cell parameters a = 155.36, b = 156.97, c = 184.66 Å. 86 521 independent reflections were obtained from the total of 357 244 measured reflections, with an R _merge value of 6.7%. The data set was 97.5% complete at the resolution limit of 2.4 Å. The crystal contained three subunits of the enzyme in the asymmetric unit. The V _M value (the crystal volume per unit protein molecular weight; Matthews, 1968 ▶) was determined to be 2.92 ų Da⁻¹ and the solvent content was 57.2% assuming the presence of three molecules of the subunit in the asymmetric unit. The V _M value and solvent content lie within the range usually found for protein crystals.

Figure 2.

Crystal of FOD_AO. The scale bar is 0.1 mm in length.

Figure 3.

X-ray diffraction pattern from a crystal of FOD. The ring indicates a resolution of 2.4 Å.

Table 1. Data-collection and processing statistics.

Beamline	SPring-8 BL-26B1
Wavelength (Å)	1.00
Detector	R-AXIS V
Crystal-to-detector distance (mm)	450
Rotation range per image (°)	0.80
Total rotation range (°)	106
Exposure time per image (s)	15.0
Size of crystal used for data collection (mm)	0.3 × 0.3 × 0.1
Resolution range (Å)	50–2.40 (2.49–2.40)
Space group	C2221
Unit-cell parameters (Å)	a = 155.36, b = 159.97, c = 184.66
Mosaicity (°)	0.578
Total No. of measured intensities	357244 (34120)
No. of independent reflections	86521 (8530)
Mean I/σ(I)	13.5 (3.7)
Multiplicity	4.1 (4.0)
Completeness (%)	97.5 (97.5)
Rmerge† (%)	6.7 (50.3)
Overall B factor from Wilson plot (Å2)	46.9
No. of molecules per asymmetric unit	3
Matthews coefficient (Å3 Da−1)	2.92
Solvent content (%)	57.2

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

All FODs isolated to date are dimeric proteins. The crystal of FOD_AO used in this report contains three subunits in the asymmetric unit. In order to obtain information about its catalytic site and modified FAD, structure determination by molecular replacement using choline oxidase (PDB code 2jbv; Quaye et al., 2008 ▶) and glucose oxidase (PDB code 1cf3; Wohlfahrt et al., 1999 ▶) is now in progress.