Overexpression, crystallization and preliminary X-­ray crystallographic analysis of shikimate dehydrogenase from Archaeoglobus fulgidus

Shikimate dehydrogenase from A. fulgidus has been overexpressed and crystallized. X-ray diffraction data have been collected to 2.8 Å resolution.

Abstract

Shikimate dehydrogenase (SDH), which catalyses the NADPH-dependent reduction of 3-dehydroshikimate to shikimate in the shikimate pathway, is an attractive target for the development of herbicides and antimicrobial agents. Previous structural studies have shown that SDH exists in two conformations, an open and a closed form, and it is believed that the conformational state is crucial to understanding its catalytic mechanism. In order to facilitate further structural comparisons among SDHs, including the conformational state, structural analysis of an SDH from Archaeoglobus fulgidus encoded by the Af2327 gene has been initiated. SeMet-labelled SDH from A. fulgidus was overexpressed in Escherichia coli and crystallized at 296 K using ammonium sulfate as a precipitant in order to use the MAD method for structure determination. Crystals of A. fulgidus SDH grown in the presence of NADP⁺ diffracted to 2.8 Å resolution and belonged to the trigonal space group P3₂21 (or P3₂21), with unit-cell parameters a = 111.3, b = 111.3, c = 76.2 Å. Three diffraction data sets were collected. The asymmetric unit contains two monomers, with a corresponding V _M of 2.34 ų Da⁻¹ and a solvent content of 47% by volume.

1. Introduction

Shikimate is the key intermediate in the shikimate-biosynthetic pathway commonly found in bacteria, fungi and higher plants but absent in animals. Therefore, the shikimate pathway is an attractive target for the development of antibacterial agents (Davies et al., 1994 ▶). The shikimate pathway consists of seven enzymatic steps. Shikimate dehydrogenase (SDH; EC 1.1.1.25), which is encoded by the aroE gene in bacteria, is responsible for the fourth reaction of the shikimate pathway. SDH catalyses the NADPH-dependent reduction of 3-­dehydroshikimate to shikimate (Singh et al., 2005 ▶). Inhibitors that target SDH from Helicobacter pylori have been reported (Han et al., 2006 ▶) and have been shown to inhibit cell growth, suggesting that SDH might be a promising target for antibacterial agents (Han et al., 2006 ▶). Two types of shikimate dehydrogenase from bacteria, SDH (PDB code 1nyt) and YdiB (PDB code 1o9b), have been structurally reported (Michel et al., 2003 ▶). Structural and biochemical studies of a novel shikimate dehydrogenase from Haemophilus influenzae revealed that it has a different substrate specificity from SDH and YdiB (Singh et al., 2005 ▶). The SDH from H. influenzae catalyzes the oxidation of shikimate but not quinate, while YdiB catalyzes the reversible reductions of dehydroquinate to quinate and of dehydro­shikimate to shikimate in the presence of NAD(P)H (Singh et al., 2005 ▶). SDH is bipartite and is composed of an NADPH-binding domain and a catalytic domain. The NADPH-binding domain has a typical Rossmann fold containing a unique glycine-rich P-loop. The catalytic domain has a novel fold (Ye et al., 2003 ▶).

The oligomeric states of SDHs, which are mostly either monomers or dimers, differ depending on species. SDH from Escherichia coli is present as a monomer, whereas SDHs usually form oligomers in most bacteria (Anton & Coggins, 1988 ▶; Chaudhuri & Coggins, 1985 ▶). Recently reported crystallographic studies of SDH from Thermotoga maritima suggest that the SDH from T. maritima exists as a monomer in solution. In comparison, SDH from Methanococcus jannaschii and YdiB from E. coli have been shown to exist as dimers in both solution and crystals (Michel et al., 2003 ▶; Padyana & Burley, 2003 ▶).

Subsequently, the structures of several SDHs have been reported, including the structures of SDHs from Arabidopsis (Singh & Christendat, 2006 ▶), Aquifex aeolicus (Gan et al., 2007 ▶), Thermus thermophilus (Bagautdinov & Kunishima, 2007 ▶) and Staphylococcus epidermidis (Han et al., 2009 ▶). Structural studies on SDHs revealed that they have two conformations: open and closed forms. The closed form has been suggested to be necessary for catalysis (Michel et al., 2003 ▶). In SDH from E. coli, substrate binding promotes a switch from the open conformation to the closed conformation (Michel et al., 2003 ▶). In SDH from T. thermophilus, it has been shown that the cofactor only binds to the closed form, while the substrate binds to both forms (Bagautdinov & Kunishima, 2007 ▶). The openness of SDHs is likely to be dependent on the species. The crystal structure of SDH from A. aeolicus in complex with NADP⁺ and shikimic acid exhibits a closed conformation (Gan et al., 2007 ▶), whereas the ternary complex of SDH from T. thermophilus with NADP⁺ and shikimic acid shows an open conformation.

In order to facilitate further structural comparisons among SDHs, including their conformation (open form or closed form), we have initiated crystallographic studies of SDH from A. fulgidus. In con­trast to bacteria, SDHs from archaea are structurally unexplored. Structural studies on SDH from A. fulgidus might facilitate the design of inhibitors targeting SDHs. When we analyzed the sequence identity of SDH from A. fulgidus to other structurally reported SDHs, the sequence identity was 33% to SDH from E. coli, 37% to YdiB from E. coli, 30% to SDH from H. influenzae, and 40% to SDH from M. jannaschii. The SDH from A. fulgidus has been overexpressed in E. coli and crystallized. Its crystallization conditions and X-ray crystallographic data are reported here.

2. Materials and methods

2.1. Protein expression and purification

The aroE gene (Af2327) encoding A. fulgidus SDH was amplified by the polymerase chain reaction. The forward and reverse oligonucleotide primers were 5′-GG GAA TTC CAT ATG CTC TAC CTT GGC GTC ATA G-3′ and 5′-CCG CCG CTC GAG TTA AAA CCT CAA AGC CCT CAA AGC AG-3′, respectively. The bases shown in bold represent the NdeI and XhoI restriction-enzyme cleavage sites, respectively. The amplified DNA was digested with NdeI and XhoI and was then inserted into the NdeI/XhoI-digested expression vector pET-21a(+) (Novagen). The plasmid was transformed into E. coli strain B834 (DE3) (Novagen) for protein expression. B834 (DE3) cells transformed with the plasmid were selected on LB–agar plates with 50 µg ml⁻¹ ampicillin. A single colony was transferred into 20 ml LB and grown overnight with vigorous shaking at 310 K. The cells were re-inoculated into 2 l M9 medium with 40 mg ml⁻¹ of all amino acids except methionine and were grown to an A ₆₀₀ of 0.5. Protein expression was induced with 1.0 mM isopropyl β-d-1-thiogalacto­pyranoside (IPTG) at 288 K. After IPTG induction, cell growth continued for 19 h at 288 K and the cells were harvested by centrifugation at 6000 rev min⁻¹ (Sorvall GSA rotor) for 10 min at 277 K. The cell pellet was resuspended in ice-cold lysis buffer A (50 mM Tris–HCl pH 7.5, 120 mM NaCl, 10 mM DTT). The cells were then homogenized by ultrasonication and heated for 10 min at 338 K. The crude cell extract was centrifuged at 36 000g (18 000 rev min⁻¹; Hanil Supra 21K rotor) for 20 min at 277 K. The supernatant was subjected to ion-exchange chromatography on a Q-Sepharose column (GE Healthcare) previously equilibrated with buffer A. Protein was eluted with a linear gradient of 0–1.0 M NaCl in buffer A. Purification was then performed by gel filtration on a HiLoad XK 16 Superdex 200 prep-grade column (GE Healthcare) previously equilibrated with buffer A containing 100 mM NaCl. The protein solution was concentrated to about 28 mg ml⁻¹ using an YM10 ultrafiltration mem­brane (Amicon). The protein concentration was estimated by measuring the absorbance at 280 nm, employing the calculated molar extinction coefficient of 21 740 M ⁻¹ cm⁻¹.

2.2. Crystallization, dynamic light scattering and X-ray data collection

Crystallization experiments were carried out using the hanging-drop vapour-diffusion method at 296 K using 24-well tissue-culture plates (Hampton Research). A hanging drop on a siliconized cover slip was prepared by mixing equal volumes (2 µl each) of protein solution and reservoir solution and was placed over 0.5 ml reservoir solution. Initial crystallization conditions were established using screening kits from Hampton Research (Crystal Screen, Crystal Screen 2 and MembFac) and deCODE Biostructures Group (Wizard I and Wizard II). Dynamic light-scattering experiments were per­formed using a DynaPro-801 instrument from Wyatt (Santa Barbara, California, USA). The data were measured at 297 K using protein at 1 mg ml⁻¹ concentration in buffer A containing 100 mM NaCl.

Crystals of A. fulgidus SDH obtained using ammonium sulfate as a precipitant were optimized. To grow crystals of the native protein complexed with NADP⁺, 100 mM NADP⁺ solution (dissolved in 50 mM Tris–HCl pH 7.5 and 100 mM NaCl) was mixed with the protein solution, resulting in an ∼50-fold molar excess of NADP⁺ over the SDH monomer. The protein mixed with NADP⁺ was incubated for 30 min at 277 K before crystallization. Crystals were flash-cooled in a liquid-nitrogen stream employing 15%(v/v) glycerol as a cryoprotectant. Three-wavelength MAD data for A. fulgidus SDH were collected at 100 K on beamline BL-18B of the Photon Factory (PF), Japan (Watanabe et al., 1995 ▶) using a MacScience 2030 image-plate detector. The wavelength of the synchrotron X-rays was 0.9792 Å for the peak, 0.9794 Å for the inflection and 0.9500 Å for the remote, and a 0.3 mm collimator was used. The crystal was rotated through a total of 180, 180 and 136° for the peak, inflection and remote data, respectively, with a 1.0° oscillation range per frame. The data set was processed and scaled using the programs MOSFLM (Leslie, 1992 ▶) and SCALA (Winn et al., 2011 ▶).

3. Results

A. fulgidus SDH was overexpressed in soluble form with a yield of ∼10 mg homogeneous protein per litre of culture. The optimized reservoir condition was 100 mM Na HEPES buffer pH 7.8, 2.2 M ammonium sulfate and 2%(v/v) polyethylene glycol (PEG) 400. Crystals in complex with NADP⁺ grew to maximum dimensions of 0.10 × 0.10 × 0.40 mm within several days (Fig. 1 ▶). Three diffraction data sets were collected using an SeMet-substituted SDH crystal. Three sets of X-ray diffraction data were collected to 2.80 Å resolution at 100 K. For the peak data set, a total of 337 367 measured reflections were merged into 13 735 unique reflections, giving an R _merge of 11.5% and a completeness of 99.9% for the peak data. The space group was determined as the trigonal space group P3₂21 (or P3₂21), with unit-cell parameters a = 111.3, b = 111.3, c = 76.2 Å on the basis of systematic absences and symmetry of diffraction intensities. Table 1 ▶ summarizes the statistics of data collection. The molecular mass of the recombinant SDH was estimated to be ∼60 kDa by dynamic light-scattering analysis, indicating that the enzyme exists as a dimer in solution (calculated monomer mass of 30 202 Da). If it is assumed that a dimeric molecule is present in the crystallographic asymmetric unit, the crystal volume per protein mass (V _M) is 2.34 ų Da⁻¹ and the solvent content is 47% (Matthews, 1968 ▶).

Figure 1.

Crystals of shikimate dehydrogenase from A. fulgidus grown in the presence of NADP⁺, with approximate dimensions of 0.2 × 0.1 × 0.4 mm.

Table 1. Data-collection and refinement statistics for SeMet-substituted A. fulgidus SDH.

Values in parentheses are for the highest resolution shell (2.95–2.80 Å).

Data	Peak	Inflection	Remote
X-ray source	BL-18B, PF	BL-18B, PF	BL-18B, PF
Wavelength (Å)	0.9792	0.9794	0.9500
Space group	P3121 (or P3221)	P3121 (or P3221)	P3121 (or P3221)
Unit-cell parameters
a = b (Å)	111.31	111.34	111.31
c (Å)	76.15	76.10	76.15
α = β (°)	90	90	90
γ (°)	120	120	120
Resolution range (Å)	32–2.8	32–2.8	32–2.8
Measured reflections	337367	337562	254186
Unique reflections	13735	13742	13733
Completeness (%)	99.9 (100)	100 (100)	99.9 (99.9)
〈I/σ(I)〉	5.7 (2.0)	5.6 (2.0)	5.2 (1.7)
Rmerge† (%)	11.5 (36.6)	11.8 (37.3)	12.9 (44.0)

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