Crystallization and preliminary X-ray analysis of the bacillaene synthase trans-acting acyltransferase PksC

The expression, purification and crystallization of the trans-acting acyltransferase PksC from the bacillaene hybrid polyketide synthase/nonribosomal peptide synthetase is described. The crystals belonged to the orthorhombic space group P2₁2₁2₁ and diffracted to 1.44 Å resolution.

Abstract

The antibiotic bacillaene is biosynthesized in Bacillus subtilis by a hybrid type 1 modular polyketide synthase/nonribosomal peptide synthetase of the trans-acyltransferase (trans-AT) class. Within this system, the essential acyl-group loading activity is provided by the action of three free-standing trans-acting acyltransferases. Here, the recombinant expression, purification and crystallization of the bacillaene synthase trans-acting acyltransferase PksC are reported. A diffraction data set has been collected from a single PksC crystal to 1.44 Å resolution and the crystal was found to belong to the orthorhombic space group P2₁2₁2₁.

1. Introduction

Polyketides (PKs), nonribosomal peptides (NRPs) and hybrids thereof (PK/NRPSs) constitute a diverse family of biologically important secondary metabolites that have found widespread application in human and veterinary medicine, agriculture and biotechnology (Walsh, 2008 ▶). Despite the astonishing structural and functional variety of these molecules, each is assembled in an analogous fashion involving the sequential addition of either organic acids or amino acids to an initial thiotemplate (Hertweck, 2009 ▶). For many products, biosynthesis is directed by the action of giant multifunctional enzymes (up to ∼5 MDa) of the type 1 modular polyketide synthase (PKS) class, in which the catalytic apparatus responsible for product assembly comprises a series of linearly arranged multi-domain extension modules, with each module responsible for the elongation of the product chain by a single acyl or amino-acid unit (Fischbach & Walsh, 2006 ▶; Khosla et al., 2009 ▶).

Historically, much of our understanding of modular synthases has derived from studies of secondary metabolic pathways from filamentous actinomyces and in particular the erythromycin-synthesizing deoxyerythromycin (DEBS) system (Katz, 2009 ▶). Recently, however, work focused on the characterization of secondary metabolic pathways from less well studied bacterial taxa has resulted in the identification of a novel group of multi-modular synthases which do not adhere to the established collinearity rules of the DEBS paradigm (Piel, 2010 ▶, and references therein). These novel PKSs, which are termed trans-­AT synthases, seem to have evolved independently from other modular systems and comprise disparate enzymatic activities of apparently unrelated metabolic origin which appear to have con­verged to yield a single functional synthase. Unlike the DEBS-type canonical cis-AT systems, in which synthase evolution is guided by the duplication and diversification of extension modules and domains therein (Jenke-Kodama et al., 2008 ▶), trans-AT systems appear to have arisen as a result of extensive horizontal gene transfer between bacteria, which accounts for their highly unusual biosynthetic frameworks, with aberrant module architectures, comprising novel catalytic domains and domain orders (Nguyen et al., 2008 ▶). As is being increasingly realised, the potent biological activities of many trans-AT PKS products makes these molecules attractive targets for clinical development (Thomas et al., 2010 ▶; Irschik et al., 2007 ▶; Hale et al., 2002 ▶).

In contrast to the canonical cis-AT modular systems, trans-AT synthases are characterized by the absence of individual acyltransferase domains within each PKS extension module, with this essential activity being provided through the action of between one and three free-standing trans-acting acyltransferases encoded within the synthase gene cluster. Trans-ATs act either as standalone functional domains, as fused tandem AT di-domains or as trans-acting enoyl reductase fusions (Cheng et al., 2009 ▶). Detailed structural and functional characterization of trans-acting acyltransferases from trans-AT PKSs will not only provide significant insight into this novel biosynthetic activity, but may also reveal routes towards the exploitation and/or manipulation of trans-acting ATs for use in the generation of derivative polyketide products with improved pharma­cological activities. Towards this goal, we have cloned, expressed and purified the trans-AT PksC from the prototypical trans-AT PKS bacillaene synthase. PksC has been subjected to crystallization screening and conditions supporting the growth of PksC crystals have been identified. A single diffraction data set to 1.44 Å resolution has been collected at Diamond Light Source.

2. Materials and methods

2.1. Gene cloning

The DNA sequence encoding PksC, a 288-amino-acid protein, was amplified from Bacillus subtilis 168 genomic DNA using the primers 5′-CATATGATCACATATGTTTTTCCA-3′ (forward) and 5′-CTC GAGTTAGCGGGGCATTGCTTC-3′ (reverse), introducing appropriate restriction sites for subsequent cloning (NdeI and XhoI sites; bold). The PCR product was introduced into the pGEMT transfer vector (Promega), from which an NdeI/XhoI fragment was excised and ligated into pre-cut pET28a (Novagen; cut with the same enzymes). The sequence of the resulting construct (pksC::pET28a), encoding an N-terminally hexahistadine-tagged variant of PksC, was verified by DNA sequencing and transformed into Escherichia coli BL21 (DE3) cells for protein expression.

2.2. Protein expression, purification and characterization

100 ml Luria–Bertani (LB) medium supplemented with 50 µg ml⁻¹ kanamycin was inoculated with a single colony of pksC::pET28a-transformed E. coli BL21 (DE3) cells and incubated overnight with shaking at 310 K. 20 ml of this culture was used to inoculate 1 l LB medium supplemented with kanamycin as above. The culture was grown until the optical density at 600 nm reached 0.6, at which point protein expression was induced by the addition of isopropyl β-d-1-thiogalactopyranoside to a final concentration of 1 mM. Following induction, the culture was grown for an additional 3–4 h before harvesting by centrifugation. The cell pellet was resuspended in His-load buffer (50 mM Na HEPES, 150 mM NaCl, 10 mM imidazole pH 7.5) supplemented with 5 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride and lysed by ultrasonication. The resulting cell lysate was clarified by centrifugation and the supernatant liquid was applied onto a pre-equilibrated (in His-load buffer) 5 ml Hi-Trap chelating column (charged with nickel; GE Healthcare). PksC was eluted with an imidazole gradient (10–500 mM) over ten column volumes. Fractions containing protein (as identified by SDS–PAGE and A _280nm) were concentrated and injected onto a Hi-Load 16/60 Superdex 75 column (GE Healthcare) pre-equilibrated in 20 mM Tris–HCl, 150 mM NaCl pH 7.5. Eluted fractions containing PksC (as identified by SDS–PAGE and and A _280nm) were pooled and con­centrated to 10 mg ml⁻¹ by ultrafiltration. The purity of the final preparation was estimated to be >95% by SDS–PAGE analysis, with a yield of approximately 5 mg protein per gram of cell paste.

2.3. Analytical ultracentrifugation

Sedimentation-velocity (SV) experiments were carried out in a Beckman Coulter (Palo Alto, California, USA) ProteomeLab XL-I analytical ultracentrifuge (AUC) using both absorbance (at 280 nm) and interference optics. All AUC runs were performed at a rotation speed of 48 000 rev min⁻¹ and an experimental temperature of 277 K. The sample volume was 400 µl for the SV experiments and the sample concentrations ranged between 1 and 5 mg ml⁻¹ (nine samples were examined in this concentration range). The partial specific volume () of the protein was calculated from its amino-acid sequence using the program SEDNTERP (Laue et al., 1992 ▶) and was extrapolated to the experimental temperature using the method described by Durch­schlag (1986 ▶). The density and viscosity of the buffer (20 mM Tris–HCl, 150 mM NaCl pH 7.5) at the experimental temperature were also calculated using SEDNTERP.

2.4. Crystallization and data collection

Initial crystallization screening was performed with commercially available screens from Qiagen using the sitting-drop vapour-diffusion technique. Crystallization experiments were set up using a Mosquito (TTP Labtech) nanolitre pipetting robot and MRC crystallization plates containing 100 µl each of the 96 conditions from the PACT screen (Newman et al., 2005 ▶). 250 nl protein solution was pipetted into the sample well of each of the 96 crystallization-plate wells followed by 250 nl reservoir solution. Plates were sealed with clear sealing film and allowed to equilibrate at 294 K. Small PksC needle crystals were observed in a single condition, PACT 74 (20% PEG 3350, 0.2 M sodium bromide, 0.1 M Bis-Tris propane pH 7.5), after ∼2 d. Subsequent optimization using the hanging-drop vapour-diffusion method at 294 K, mixing 1 µl protein solution with 1 µl reservoir solution, resulted in the growth of large PksC crystal clusters comprising multiple rod-shaped crystals in crystallization solutions comprising 17–22% PEG 3350, 0.2 M sodium bromide, 0.1 M bis-tris propane pH 9.5 that achieved maximum dimensions (50 × 600 µm) in around one week. Single rod-shaped PksC crystals were separated from the clusters using an acupuncture needle, cryoprotected in well solution supplemented with 25% glycerol and flash-frozen in liquid nitrogen. Diffraction data were collected at Diamond Light Source, England (station I04 equipped with an ADSC Quantum 315r CCD detector). Crystals were maintained at cryogenic temperature during data collection. All data were processed with MOSFLM (Leslie, 2006 ▶) and scaled using SCALA (Evans, 2006 ▶) as implemented in the CCP4 suite of programs (Collaborative Com­putational Project, Number 4, 1994 ▶).

3. Results and discussion

An N-terminally hexahistadine-tagged version of the bacillaene synthase trans-acyltransferase PksC has been successfully cloned and recombinantly overexpressed in E. coli. Soluble recombinant PksC has been purified to homogeneity using a combination of nickel-affinity His-chelation and size-exclusion chromatography (Fig. 1 ▶). Initial characterization by analytical ultracentrifugation revealed this material to be a monodisperse single species with a native molecular mass of 35.8 kDa (Fig. 1 ▶), implying that PksC is monomeric in solution. Diffraction-quality crystals were grown using the hanging-drop vapour-diffusion method with a well solution consisting of 17–22% PEG 3350, 0.2 M sodium bromide, 0.1 M Bis-Tris propane pH 9.5 (Fig. 2 ▶). A single X-ray diffraction data set was collected on beamline I04 at Diamond Light Source, with the crystal yielding good diffraction images. Crystal parameters and diffraction data statistics are summarized in Table 1 ▶. The crystal belonged to the orthorhombic space group P2₁2₁2₁, as determined using the autoindexing routine of MOSFLM (Leslie, 2006 ▶) and SCALA (Evans, 2006 ▶) from the CCP4 package (Collaborative Computational Project, Number 4, 1994 ▶) and inspection of systematic absences, with unit-cell parameters as given in Table 1 ▶. The asymmetric unit contained one molecule of PksC, giving a crystal volume per protein mass (V _M) of 2.07 ų Da⁻¹ and a solvent content of 40.6% (Matthews, 1968 ▶). A complete structure determination of PksC is currently under way in order to provide insight into the catalytic activity and substrate specificity of this enzyme.

Figure 1.

(a) 15% SDS–PAGE analysis of recombinant purified PksC. Molecular-weight markers are annotated in kDa. (b) Size-distribution [c(s)] analysis of PksC sedimentation-velocity data converted to standard conditions.

Figure 2.

Photomicrograph of PksC crystals.

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

Values in parentheses are for the highest resolution shell.

Data collection
X-ray source	I04, Diamond Light Source
Wavelength (Å)	0.9699
Temperature (K)	100
Detector	ADSC Quantum 315r CCD
Space group	P212121
Unit-cell parameters (Å)	a = 53.19, b = 70.43, c = 76.28
Resolution range (Å)	37.09–1.44 (1.51–1.44)
VM (Å3 Da−1)	2.07
Solvent content (%)	40.6
Monomers in asymmetric unit	1
Data processing
No. of observed reflections	354490
No. of unique reflections	53023
Completeness (%)	99.9 (99.9)
Mosaicity (°)	0.32
Multiplicity	6.7 (6.7)
Rmerge† (%)	7.1 (30.6)
Mean I/σ(I)	16.6 (5.5)

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