Crystallization and preliminary X-ray analysis of isopentenyl diphosphate isomerase from Methanocaldococcus jannaschii

Isopentenyl diphosphate isomerase from M. jannaschii has been overexpressed in E. coli, purified and crystallized. Diffraction data were collected to 2.08 Å resolution.

Abstract

Type 2 isopentenyl diphosphate isomerase (IDI-2) is a flavoprotein. Recently, flavin has been proposed to play a role as a general acid–base catalyst with no redox role during the enzyme reaction. To clarify the detailed enzyme reaction mechanism of IDI-2 and the unusual role of flavin, structural analysis of IDI-2 from Methanocaldococcus jannaschii (MjIDI) was performed. Recombinant MjIDI was crystallized at 293 K using calcium acetate as a precipitant. The diffraction of the crystal extended to 2.08 Å resolution at 100 K. The crystal belonged to the tetragonal space group I422, with unit-cell parameters a = 126.46, c = 120.03 Å. The presence of one monomer per asymmetric unit gives a crystal volume per protein weight (V _M) of 3.0 ų Da⁻¹ and a solvent constant of 59.0% by volume.

1. Introduction

Isoprenoid compounds are ubiquitous in all living species and are diverse in biological function. All isoprenoids are synthesized from the key C₅ units isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In terpenoid biosynthesis, IPP serves as the elongating C₅ donor and isomerizes to DMAPP. Isopentenyl diphos­phate isomerase (IDI; EC 5.3.3.2) is responsible for the isomerization of the carbon–carbon double bond of IPP, forming the potent electrophile DMAPP (Koyama & Ogura, 1999 ▶).

IDIs are classified into two distinct types that show no sequence homology to each other. Type 1 IDI (IDI-1) is utilized in most eukaryotes, some bacteria and some halophilic archaea, whereas type 2 IDI (IDI-2) has been reported to be produced by bacteria such as streptomycetes, bacilli and cyanobacteria, trypanosomatids and other archaea (Kuzuyama & Seto, 2003 ▶).

IDI-1 is a compact globular α/β protein and requires two divalent metal cations such as Mg²⁺ and Zn²⁺ for activity (Lee & Poulter, 2006 ▶). In contrast, IDI-2 is a flavoprotein and requires a divalent cation such as Mg²⁺ for activity. The overall chemical transformation catalyzed by IDI involves a net 1,3 proton-addition/elimination reaction. Interestingly, IDI-2 requires reduced FMN to carry out this redox-neutral isomerization (Bornemann, 2002 ▶). IDI-2 reconstituted with cofactor analogues such as 5-deaza-FMN showed no activity, suggesting that FMN plays more than a structural role (Hemmi et al., 2004 ▶; Kittleman et al., 2007 ▶). Previously, we showed that 3,4-epoxy-3-­methylbutyl diphosphate (EIPP), which has been reported to be an IDI-1 mechanism-based inhibitor, can irreversibly inhibit IDI-2 from Methanocaldococcus janaschii (MjIDI) and that it covalently binds to the N5 position of reduced FMN instead of to the active-site cysteine of IDI-1 (Hoshino et al., 2006 ▶). Furthermore, Rothman and coworkers showed that epoxy, diene and fluorinated substrate analogues give similar results (Rothman et al., 2008 ▶). These results indicate that the isomerization mechanism of IDI-2 is a protonation/deprotonation mechanism via a carbocation intermediate similar to that of IDI-1 and that the N5 position of reduced FMN plays a key role in the enzyme reaction. This mechanism has also been supported by measurement of the deuterium kinetic isotope effect (Thibodeaux et al., 2008 ▶) and inhibition study by using alkyne and allene substrate analogues (Sharma et al., 2010 ▶).

Crystal structures of IDI-2 from Bacillus subtilis (Steinbacher et al., 2003 ▶), Thermus thermophilus (de Ruyck et al., 2008 ▶) and Sulfolobus shibatae (Unno et al., 2009 ▶) revealed that IDI-2 has a TIM-barrel structure and that its active site is located inside the barrel. From structures of IDI-2 complexed with substrates and the results of kinetic analyses of IDI-2 mutants containing amino-acid substitutions in the active site, Unno and coworkers proposed that the reduced FMN plays the role of an donor/acceptor in the protonation/deproton­ation mechanism and that IDI-2 is the first reported flavoenzyme in which reduced flavin acts as a general acid–base catalyst without any redox function. Recently, by synthesizing several FMN analogues and investigating their effects on kinetic parameters, Thiobodeaux and coworkers concluded that the reduced FMN functions as an acid–base catalyst and proposed that the N5 atom of the FMN is one of the acid–base groups that are required for enzymatic turnover (Thibodeaux et al., 2010 ▶). However, the other acid–base groups were not identified; therefore, several mechanistic pathways are still being discussed.

We have undertaken the structural analysis of MjIDI and its mechanism-based inhibitor, which will provide detailed information on the mechanism of IDI-2 when combined with the results of a previous kinetic study. In this study, we report the crystallization and preliminary X-ray diffraction analysis of MjIDI.

2. Materials and methods

2.1. Expression and purification

MjIDI is composed of 359 amino acids and its molecular weight is 40 kDa. Its expression in Escherichia coli was carried out in accordance with a previously described method (Hoshino et al., 2006 ▶). Purification was performed using the previously described method with minor modifications. After heat-treatment of the cell lysate, the supernatant was loaded onto a DEAE Sepharose Fast Flow (GE Healthcare) column previously equilibrated with 20 mM HEPES pH 7.5 at a flow rate of 1 ml min⁻¹; the column was eluted with a linear NaCl con­centration gradient from 0.2 to 0.6 M. The fractions con­taining MjIDI were combined and concentrated to approximately 2 ml by centrifugation with a Centriprep filter (Amicon Inc.). The concentrate was then loaded onto a 1 ml RESOURCE PHE column (GE Healthcare) previously equilibrated with 20 mM HEPES pH 7.5 containing 1 M ammonium sulfate and the column was washed with ten bed volumes of the same buffer. The adsorbed proteins were then eluted with 20 mM HEPES pH 7.5 containing a linear gradient of ammonium sulfate (1–0 M) for 30 min at 1 ml min⁻¹. MjIDI eluted in fractions containing approximately 0.3 M ammonium sulfate. The purified enzyme was buffer-exchanged into 50 mM HEPES pH 7.5, concentrated to 10 mg ml⁻¹ and stored at 193 K.

2.2. Crystallization

Crystallization conditions for MjIDI were initially screened by the sitting-drop vapour-diffusion method at 293 K using the commercially available sparse-matrix screening kits Crystal Screen, Crystal Screen 2, Crystal Screen Cryo, PEG/Ion Screen (Hampton Research), Wizard I, Wizard II, EBS Cryo I, EBS Cryo II (Emerald Bio­Structures), Structure Screen 1 and Structure Screen 2 (Molecular Dimensions). 1 µl protein solution was mixed with an equal volume of reservoir solution and equilibrated against 100 µl reservoir solution. Layered and stacked crystals appeared using EBS Cryo II condition No. 33 [45%(v/v) glycerol, 0.1 M sodium cacodylate pH 6.5 and 0.2 M calcium acetate]. Crystals that diffracted well were obtained using a reservoir consisting of 0.6 M calcium acetate and 50 mM HEPES pH 7.5 and grew to maximum dimensions of approximately 0.10 × 0.05 × 0.05 mm in 1 d (Fig. 1 ▶).

Figure 1.

Typical appearance of crystals in hanging drops. The maximum dimensions of the crystals are approximately 0.1 × 0.05 × 0.05 mm.

2.3. X-ray diffraction study

Crystals were soaked for a few seconds in a solution consisting of 0.6 M calcium acetate and 25%(w/v) glycerol. The crystal was mounted in a cryoloop and flash-cooled in a liquid-nitrogen stream at 100 K. X-­ray diffraction data were collected from MjIDI crystals on beamline BL38B1 at SPring-8 using an ADSC Quantum 210 CCD detector system (ADSC, California, USA). Data collection was performed at a wavelength of 1.0 Å with a total oscillation range of 100°. Each diffraction image was obtained with an oscillation angle of 0.5° and an exposure time of 5.0 s. The crystal-to-detector distance was set to 150 mm. The data were integrated and scaled using the HKL-2000 program package (Otwinowski & Minor, 1997 ▶).

3. Results

Single crystals of recombinant MjIDI were obtained by the sparse-matrix method and were optimized to produce single crystals that were suitable for X-ray analysis. Crystals grew to maximum dimensions of 0.1 × 0.05 × 0.05 mm (Fig. 1 ▶). X-ray diffraction data for MjIDI were collected to a resolution of 2.08 Å from flash-cooled crystals on the BL38B1 beamline at SPring-8 (Fig. 2 ▶). From the diffraction data collected, the space group was determined to be tetragonal I422, with unit-cell parameters a = 126.46, c = 120.03 Å. The number of molecules in the asymmetric unit was estimated from the Matthews probability (Kantardjieff & Rupp, 2003 ▶). The highest probability (0.99) was obtained for one monomer in the asymmetric unit; the estimated Matthews coefficient was 3.0 ų Da⁻¹, corresponding to a solvent content of 59.0%(v/v). Scaling and merging of the crystallo­graphic data resulted in an overall R _merge of 6.7%. Complete data-collection statistics are shown in Table 1 ▶.

Figure 2.

Diffraction pattern of an MjIDI crystal. An enlarged image of the area around 2.08 Å resolution is shown.

Table 1. Data-collection and diffraction data statistics.

Values in parentheses are for the highest resolution shell.

X-ray source	SPring-8 BL38B1
Detector	ADSC Quantum 210
Wavelength (Å)	1.0000
Crystal-to-detector distance (mm)	150
Oscillation angle (°)	0.5
Total oscillation range (°)	100
Space group	I422
Unit-cell parameters (Å)	a = 126.46, c = 120.03
Resolution (Å)	35.00–2.08 (2.15–2.08)
Multiplicity	7.9 (7.8)
Completeness (%)	99.8 (100)
Rmerge† (%)	6.7 (42.6)
〈I/σ(I)〉	34.6 (6.2)

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

The structure was solved by the molecular-replacement method using the MOLREP program (Vagin & Teplyakov, 2010 ▶) in the CCP4 suite (Collaborative Computational Project, Number 4, 1994 ▶) with the rigid-body refinement option. X-ray diffraction data from 20 to 2.3 Å resolution were used in this step. The 2.0 Å resolution X-ray structure of monomer A of substrate-free isopentenyl diphosphate isomerase from S. shibatae (PDB code 2zru; Unno et al., 2009 ▶) was used as a search model; the sequence homology between the target protein and the model was 42%, which is higher than those for the other structurally known IDI-2s (i.e. those from B. subtilis and T. thermophilus, which have 28 and 29% sequence homology, respectively). Water molecules and other ions were removed from the model. The model was subsequently subjected to rigid-body refinement in REFMAC5 (Murshudov et al., 1997 ▶) within the CCP4 package, giving an R factor of 36.2%. Further model building and restrained refinement are currently in progress.