Purification, crystallization and preliminary X-ray diffraction analysis of Omp6, a protoilludene synthase from Omphalotus olearius

The expression, purification and crystallization of Omp6, a protoilludene synthase from O. olearius, are described.

Abstract

Basidiomycetes produce a wide range of industrially relevant natural products. One of the main classes of natural products isolated from fungi are terpenoids, a highly diverse group of secondary metabolites, many of which are bioactive and have been adapted for pharmaceutical purposes. The discovery of a suite of novel sesquiterpene synthases from Omphalotus olearius via genome sequencing and bioinformatic analyses has recently been described. Here, the expression, purification and crystallization of one of these enzymes (Omp6), a protoilludene synthase, is reported. A native crystal diffracted to a resolution of 2.9 Å and belonged to space group P2₁, with unit-cell parameters a = 43.67, b = 76.76, c = 107.22 Å, α = γ = 90, β = 95°. A diffraction data set was collected on a home-source Rigaku/MSC MicroMax-007 X-ray generator.

1. Introduction  

Basidiomycetes are a rich resource for many industrially relevant natural products owing to the fact that they produce an extremely diverse range of secondary metabolites (Wasser, 2011 ▶). The unique properties of the chemical structures and bioactivities of these secondary metabolites have enabled the robust survival and evolv­ability of fungal species. Notably, many of these bioactive compounds have been isolated and adapted for pharmaceutical purposes, including as antimicrobial, anti-inflammatory and anticancer drugs (Zhong & Xiao, 2009 ▶).

The main type of bioactive secondary metabolite that has been isolated from Basidiomycetes is a class of compounds known as terpenoids, in particular the sesquiterpenes (Abraham, 2001 ▶). Sesquiterpenes are produced by a family of enzymes called sesquiterpene synthases. These enzymes cyclize the 15-carbon molecule farnesyl pyrophosphate (FPP), yielding several hundred different types of volatile cyclic hydrocarbon backbones (Miller & Allemann, 2012 ▶; Christianson, 2006 ▶). Despite this wide product diversity, all sesquiterpene synthases share a common three-dimensional fold and a catalytic mechanism involving the metal-ion-mediated dephos­phorylation of FPP to produce a reactive carbocation. This carbocation undergoes a series of rearrangement reactions until a final quenching by water, releasing the final hydrocarbon product from the catalytic active site (Yamada et al., 2012 ▶).

We recently described the genome sequencing, genome mining and subsequent discovery of a suite of novel sesquiterpene synthases from Omphalotus olearius, a Basidiomycete which had previously been shown to produce the anticancer sesquiterpene scaffold illudin (McMorris et al., 1990 ▶, 2002 ▶; Wawrzyn et al., 2012 ▶). Two of these novel sesquiterpene synthases, Omp6 and Omp7, have been shown to be highly active and product-specific protoilludene synthases, producing Δ-6 protoilludene via a likely 1,11 ring closure of FPP to create a trans-humulyl cation intermediate followed by two cyclization steps to yield the final volatile sesquiterpene (Fig. 1 ▶; Wawrzyn et al., 2012 ▶). Recent studies have shown that Δ-6 protoilludene is also produced by active-site mutants of the closely related pentalenene synthase via premature deprotonation of the carbocation. These data strongly suggest that protoilludene synthases and pentalenene synthases share a common reaction mechanism (Zu et al., 2012 ▶; Seemann et al., 2002 ▶; Lesburg et al., 1997 ▶). However, despite major attempts to alter the product-specifity of the protoilludene synthases from O. olearius via site-directed mutagenesis and via varying the reaction conditions, we have not observed these enzymes producing significant amounts of pentalenene. Therefore, in order to fully understand not only the cyclization mechanism but also the product-specificity of protoilludene synthase, we set out to crystallize and solve the three-dimensional structure of this enzyme. Here, we report the expression, purification, crystallization and preliminary X-ray analysis of the 357-­amino-acid 41 068 Da Omp6 protoilludene synthase, the crystal structure of which would be the first of a sesquiterpene synthase that displays this activity.

Figure 1.

Proposed cyclization pathway leading to Δ-6 protoilludene. A 1,11 ring closure of the carbocation from (E,E)-FPP would yield a trans-humulyl cation intermediate. Hydride shifts and two cyclization steps would result in the hydrocarbon Δ-6 protoilludene. OPP represents the diphosphate moiety of the 15-carbon compound.

2. Methods  

2.1. Overexpression and purification of Omp6  

Omp6 was PCR-amplified using Omp6 which had previously been cloned into our in-house pUCBB vector as a template (Vick et al., 2011 ▶; Wawrzyn et al., 2012 ▶) using the high-fidelity Pfu polymerase (Roche). The oligonucleotides Omp6_FWD, 5′-TAGGATCCATGATTGCAAAGAACTCCG-3′, and Omp6_REV, 5′-TAGCGGCCGCTTAAGTACTTTGAGC-3′, were used to yield a PCR product with BamHI and NotI restriction sites (shown in bold). The 1074 bp gene was cloned into the pET32b vector (EMD Millipore) and the resulting Omp6-pET32b plasmid was verified by DNA sequencing (ACGT Inc.). This construct enables the expression of Omp6 with an N-­terminal fusion thioredoxin (Trx) tag, which can aid in the soluble expression of proteins (LaVallie et al., 2003 ▶), as well as an N-terminal His₆ fusion for purification purposes. The linker region between Trx and Omp6 contains an enterokinase-specific cleavage site, allowing the separation of Omp6 from the 135-amino-acid fusion-tag region.

Omp6-pET32b was transformed into Escherichia coli C2566 cells (NEB) and single colonies were isolated on lysogeny broth (LB) agar supplemented with 100 µg ml⁻¹ ampicillin. A single colony was used to inoculate a 50 ml LB overnight culture supplemented with 100 µg ml⁻¹ ampicillin, which was incubated at 303 K. This culture was used to inoculate 3 l LB supplemented with 100 µg ml⁻¹ ampicillin, which was incubated at 303 K until mid-log phase (OD₆₀₀ = 0.6). Expression of Omp6 was induced by adding a final concentration of 0.5 mM IPTG. The cells were grown for a further 2.5 h at 303 K and were harvested by centrifugation.

The cells were resuspended in buffer A (20 mM Tris–HCl pH 8.0, 200 mM NaCl, 10 mM MgCl₂, 5 mM imidazole) and were lysed by sonication. The soluble protein was separated from the cell slurry by centrifugation at 15 000g for 30 min at 277 K and the supernatant was clarified by passage through a 0.45 µm filter device (EMD Millipore). The protein was loaded onto a 5 ml HisTrap FF column (GE Healthcare) pre-equilibrated with buffer A. The His₆-tagged protein was eluted with buffer B (20 mM Tris–HCl pH 8.0, 200 mM NaCl, 10 mM MgCl₂, 250 mM imidazole) and the Trx and His₆ tags were subsequently removed from the Omp6 by overnight incubation with enterokinase (EMD Millipore). The cleaved Trx and His₆ tags were separated from Omp6 by passage over a 5 ml HisTrap FF column and Omp6 was further purified by gel filtration using a Superdex 200 10/300 GL size-exclusion column pre-equilibrated with buffer C (20 mM Tris–HCl pH 8.0, 200 mM NaCl, 10 mM MgCl₂, 1 mM β-mercapto­ethanol). The purity of the Omp6 was verified by SDS–PAGE.

2.2. Enzyme assays  

The activity of the purified Omp6 was confirmed using gas chromatography–mass spectrometry (GC-MS). In vitro assays were performed in buffer C in a final reaction volume of 100 µl. Omp6 (10 µg) was incubated with 100 µM (E,E)-FPP in a sealed glass vial and reactions were carried out at 298 K for 18 h. The headspace of the reaction vessel was sampled for 10 min by solid-phase microextraction (SPME) using a 100 µm polydimethylsiloxane fiber (Supelco, Bellefonte, Pennsylvania, USA). The fiber was inserted into the injection port of a GC-MS for desorption of compounds, and analysis was conducted on an HP GC 7890A coupled to an anion-trap mass spectrometer HP MSD triple-axis detector (Agilent Technologies, Santa Clara, California, USA). Separation of the compounds was performed using an HP-5MS capillary column (30 m × 0.25 mm × 1.0 µm) with an injection-port temperature of 523 K and helium as a carrier gas. The oven temperature started at 333 K and was increased by 10 K min⁻¹ to a final temperature of 523 K. Mass spectra were scanned in the range 5–300 atomic mass units at 1 s intervals. The compounds produced were identified by first calibrating the GC-MS with a C₈–C₂₀ alkane mixture. The retention indices and mass spectra of compound peaks were compared with reference spectra in MassFinder 4.

2.3. Crystallization of Omp6  

Purified Omp6 was concentrated to 10 mg ml⁻¹ in 20 mM Tris–HCl pH 8.0, 200 mM NaCl, 10 mM MgCl₂, 1 mM β-mercaptoethanol for initial crystallization trials. Crystallization was carried out using the sitting-drop vapor-diffusion technique. Microplate trials were set up using a Rigaku CrystalMation System at the Nanoliter Crystallization Facility at the University of Minnesota. The commercial screens JCSG+ and PACT Suite (Qiagen) were used to set up 100 µl wells in microplates across 192 conditions. 100 nl protein was pipetted into each well with 100 nl crystallant. Plates were sealed with clear sealing film and were allowed to equilibrate at 277 K. Omp6 crystals were observed after 2 d as small needles of dimensions 10 × 5 × 2 µm in PACT Suite condition B12 [0.01 M ZnCl₂, 20%(w/v) PEG 6000, 0.1 M MES pH 6.0]. Manual screens were set up with this condition in 24-well trays using the hanging-drop vapor-diffusion technique with 1 ml crystallant in the reservoir and drops consisting of 1 µl protein mixed with 1 µl crystallant. The trays were allowed to equilibrate at 277 K. Crystals appeared in 2 d from drops consisting of 0.01 M ZnCl₂, 20%(w/v) PEG 6000, 0.1 M MES pH 6.0 and grew to final dimensions of 100 × 50 × 20 µm.

2.4. Data collection and processing  

The crystals were transferred into a cryoprotectant consisting of 0.01 M ZnCl₂, 20%(w/v) PEG 6000, 0.1 M MES pH 6.0, 1%(w/v) sucrose and were mounted in a 0.1 mm CryoLoop (Hampton Research) before flash-cooling by immediate immersion into liquid nitrogen at 100 K.

Diffraction data were collected to a resolution of 2.9 Å using a wavelength of 1.54 Å on a Rigaku/MSC MicroMax-007 X-ray generator at the Kahlert Structural Biology Laboratory at the University of Minnesota. A total of 264 images were collected with an oscillation angle of 0.5° and an exposure time of 90 s per image. The data were indexed using iMOSFLM (Battye et al., 2011 ▶) and were scaled using SCALA in CCP4 (Winn et al., 2011 ▶; Evans, 2006 ▶). Data-collection and processing statistics are summarized in Table 1 ▶.

Table 1. Data-collection and processing statistics for Omp6.

Values in parentheses are for the outermost resolution shell.

Data collection
Space group	P21
Unit-cell parameters (Å, °)	a = 43.67, b = 76.76, c = 107.22, α = γ = 90, β = 95
Wavelength (Å)	1.54
Resolution (Å)	23.14–2.90 (3.06–2.90)
Matthews coefficient V M (Å3 Da−1)	2.23
Solvent content (%)	44.77
Monomers in asymmetric unit	2
Data processing
No. of observed reflections	36143
No. of unique reflections	14699
Completeness (%)	94.0 (85.7)
Multiplicity	2.5 (1.8)
R merge † (%)	11.2 (22.7)
Mean I/σ(I)	6.4 (2.7)

^† R _merge = , where I _i(hkl) is the ith observation of reflection hkl and 〈I(hkl)〉 is the weighted average intensity for all observations i of reflection hkl.

3. Results and discussion  

Recombinant Omp6 from O. olearius was expressed as a soluble fusion with Trx in E. coli C2566 cells. The protein was purified to near-­homogeneity by Ni-affinity chromatography and size-exclusion chromatography, resulting in a yield of 1.5 mg purified protein from 3 l culture. SDS–PAGE analysis revealed a single band corresponding to a molecular weight of about 40 kDa, which is close to the calculated molecular weight of 41.07 kDa. Omp6 eluted from a Superdex 200 size-exclusion column as a single peak at 15.2 ml, suggesting that it behaves as a monomer in solution, while contaminating proteins eluted in the void volume (8.0 ml) (Fig. 2 ▶). Enzyme assays carried out using the purified protein confirmed Omp6 to be a highly active and specific protoilludene synthase (Fig. 3 ▶).

Figure 2.

Purification of Omp6 by size-exclusion chromatography. The chromatogram shows two main peaks; SDS–PAGE (15%) analysis of the proteins contained within these fractions shows that the peak at the void volume (8.0 ml) contains unwanted proteins, while the 41 kDa Omp6 (highlighted with an arrow) elutes as a distinct peak (15.2 ml) from the Superdex 200 column.

Figure 3.

Activity of purified Omp6. GC-MS analysis of an in vitro assay of Omp6 incubated with (E,E)-FPP. The enzyme produces Δ-6 protoilludene as its sesquiterpene product.

Crystals of Omp6 were obtained using 0.01 M ZnCl₂, 20%(w/v) PEG 6000, 0.1 M MES pH 6.0. Initial high-throughput crystallization screens produced crystals that were deemed to be too small to mount in a X-ray beam. The crystallization conditions were successfully reproduced manually using the hanging-drop technique, allowing the growth of Omp6 crystals to dimensions of 100 × 50 × 20 µm (Fig. 4 ▶). While these crystals were still reasonably small, it was possible to harvest them and to test them for diffraction quality. The best diffraction was obtained from crystals which were cryoprotected in mother liquor supplemented with 1%(w/v) sucrose. It was found that the choice of cryoprotectant was important in obtaining suitable diffraction; screening of other cryoprotectants at a range of concentrations, including PEG 400, glucose and glycerol, yielded diffraction to 6 Å resolution at best. Upon soaking several Omp6 crystals in gradually decreasing concentrations of sucrose, the resolution limits of diffraction of each crystal gradually increased. A crystal soaked in cryoprotectant supplemented with 1%(w/v) sucrose diffracted to 2.9 Å resolution (Fig. 5 ▶), allowing a complete native data set to be collected.

Figure 4.

Crystals of Omp6. The scale bar represents 100 µm.

Figure 5.

Diffraction pattern from a single Omp6 crystal. The crystal diffracted to a resolution of 2.9 Å.

The Omp6 crystal belonged to the monoclinic space group P2₁, with unit-cell parameters a = 43.67, b = 76.76, c = 107.22 Å, α = γ = 90, β = 95°. Assuming the presence of two molecules in the asymmetric unit, the calculated Matthews coefficient and solvent content are 2.23 ų Da⁻¹ and 44.77%, respectively (Matthews, 1968 ▶). Attempts to solve the structure of Omp6 by molecular replacement with Phaser (McCoy et al., 2007 ▶) and MOLREP (Vagin & Teplyakov, 2010 ▶) using PDB entries 3kb9 (24% sequence identity; Aaron et al., 2010 ▶) and 1ps1 (26% sequence identity; Lesburg et al., 1997 ▶) as search templates were unsuccessful. Further crystallization trials are currently under way with the aim of producing crystals of suitable diffraction quality to allow SAD phasing. Solution of the structure of Omp6 would represent the first crystal structure of a protoilludene synthase and would provide key information about the catalytic mechanism of this industrially relevant enzyme.