Search tips
Search criteria 


Logo of actafjournal home pagethis articleInternational Union of Crystallographysearchsubscribearticle submission
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 April 1; 64(Pt 4): 304–306.
Published online 2008 March 29. doi:  10.1107/S1744309108006775
PMCID: PMC2374239

Crystallization and preliminary crystallographic analysis of a plant type III polyketide synthase that produces benzalacetone


Benzalacetone synthase (BAS) from Rheum palmatum is a plant-specific type III polyketide synthase that catalyzes the one-step decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce the diketide 4-(4-hydroxyphenyl)-but-3-en-2-one. Recombinant BAS expressed in Escherichia coli was crystallized by the sitting-drop vapour-diffusion method. The crystals belong to space group P21, with unit-cell parameters a = 54.6, b = 89.6, c = 81.1 Å, α = γ = 90.0, β = 100.5°. Diffraction data were collected to 1.8 Å resolution using synchrotron radiation at BL24XU of SPring-8.

Keywords: benzalacetone synthases, polyketide synthases, chalcone synthases

1. Introduction

Benzalacetone synthase (BAS; 384 amino acids; 42.2 kDa per monomer), a plant-specific chalcone synthase (CHS) superfamily enzyme of the type III polyketide synthases (PKSs), is thought to play a crucial role in the construction of the C6–C4 moiety of a variety of pharmaceutically important phenylbutanoids, including the anti-inflammatory glucoside lindleyin from the medicinal plant Rheum palmatum (Abe et al., 2001 [triangle]). Although many of the type III PKSs catalyze iterative condensations of malonyl-CoA with a starter sub­strate, BAS catalyzes the one-step decarboxylative condensation with 4-coumaroyl-CoA to produce the diketide benzalacetone 4-(4-hydroxyphenyl)-but-3-en-2-one (Fig. 1 [triangle] a). The diketide-producing BAS obtained from R. palmatum shares ~70% amino-acid sequence identity with the regular CHS (EC, which produces the tetraketide naringenin chalcone from 4-coumaroyl-CoA after three condensations with malonyl-CoA (Fig. 1 [triangle] b). Recent crystallographic and site-directed mutagenesis studies of several type III PKSs have begun to reveal that only a small modification of the active-site architecture generates the remarkable functional diversity of the CHS-superfamily enzymes (Ferrer et al., 1999 [triangle]; Jez, Austin et al., 2000 [triangle]; Austin et al., 2004 [triangle]; Morita et al., 2007 [triangle]). In R. palmatum BAS, the conserved active-site residues of CHS, Phe208 and Thr190 (corresponding to Phe215 and Thr197 in Medicago sativa CHS), are uniquely replaced by Leu and Cys, respectively. The conformationally flexible Phe208, which is located at the junction between the active-site cavity and the CoA-binding tunnel, is strictly conserved in all known type III PKSs and has been proposed to facilitate the decarboxylation of malonyl-CoA and to help orient substrates/intermediates during the sequential condensation reaction (Jez, Ferrer et al., 2000 [triangle]). On the other hand, the chemically inert Thr190, which is altered in a number of divergent type III PKSs, has been shown to be important for steric modulation of the active-site cavity, thereby controlling the poly­ketide chain length and the product specificity (Jez, Austin et al., 2000 [triangle]; Abe et al., 2005 [triangle]). We previously demonstrated that replacement of Leu208 by Phe in R. palmatum BAS is responsible for the interruption of the polyketide-chain elongation at the diketide stage, while mutation of Cys190 to Thr did not significantly affect the diketide-forming activity of R. palmatum BAS (Fig. 1 [triangle] b; Abe et al., 2003 [triangle], 2007 [triangle]). Furthermore, our homology modelling studies suggested that unlike the case of CHS, R. palmatum BAS utilizes an alternative active-site pocket to lock the aromatic moiety of the coumaroyl starter substrate for the diketide-formation reaction (Abe et al., 2007 [triangle]). To test this hypothesis and to further clarify the intimate three-dimensional structural details of the enzyme-catalyzed processes, we carried out a crystallographic analysis of R. palmatum BAS.

Figure 1
Proposed pathways for the formation of (a) 4-(4-hydroxyphenyl)-but-3-en-2-one by BAS and (b) naringenin chalcone by CHS.

2. Experimental

2.1. Expression and purification

The pET22b(+) vector encoding full-length BAS (Abe et al., 2001 [triangle]) was used as the template to amplify the BAS gene by PCR, using 5′-­GCGCCCGGGAATGGCAACTGAGGAGATGAAGAAATTGGC-3′ as the sense primer, which introduces a SmaI restriction site, and 5′-GCGGTCGACCTAGCTAATTACGGGCACACTGCGTA­GCA-3′ as the antisense primer, which introduces a SalI restriction site. The amplified DNA fragment was digested with SmaI/SalI and cloned into the SmaI/SalI sites of a modified pQE81L expression vector (Qiagen) for expression as a glutathione S-transferase (GST) fusion protein at the N-terminus. Between GST and BAS, a Pre­Scission Protease (GE Healthcare) cleavage site (LEVLFQGP) was introduced. After confirmation of the sequence, the resultant expression plasmid was transformed into Escherichia coli M15. The cells harbouring the plasmid were cultured to an OD600 of 0.6 in LB medium containing 50 µg ml−1 carbenicillin at 303 K. Isopropyl β-d-1-thiogalactopyranoside was added to a final concentration of 0.4 mM to induce gene expression and the culture was incubated for a further 16 h at 303 K.

All of the following procedures were performed at 277 K. The E. coli cells were harvested by centrifugation at 5000g and resuspended in 50 mM Tris–HCl buffer pH 8.0 containing 0.2 M NaCl, 5%(v/v) glycerol and 2 mM DTT (buffer A). The cells were disrupted by sonication and the lysate was centrifuged at 12 000g for 30 min. The supernatant was loaded onto a Glutathione Sepharose 4B affinity column (GE Healthcare) equilibrated with buffer A and the column was then washed with buffer A. The GST tag was cleaved on the column by PreScission Protease overnight and the recombinant BAS was eluted with buffer A. The resultant BAS protein thus contains three additional residues (Gly-Pro-Gly) at the N-terminal flanking region derived from the PreScission Protease recognition sequence. After fivefold dilution of the protein solution with 50 mM Tris–HCl buffer pH 8.0 containing 5%(v/v) glycerol and 2 mM DTT, the protein solution was passed through a CIM-SO3-8 monolithic column (BIA Separations) and the flowthrough fraction was collected. The protein solution was further purified to homogeneity by chromatography on a Superdex 200HR (10/300GL) column (GE Healthcare) and was concentrated to 20 mg ml−1 in 20 mM HEPES–NaOH pH 7.5 buffer containing 100 mM NaCl and 2 mM DTT. The typical yield of protein was about 10 mg per litre of culture.

Dynamic light-scattering (DLS) analysis was carried out using a DynaProMSXTC molecular-sizing instrument (Protein Solutions Inc.). After centrifugation with a 0.22 µm Ultrafree-MC filter (Millipore) to remove particulate material from the protein solution, the solution properties of the purified protein were monitored. The data were acquired from 50 scattering measurements at 278 K; the data from five sets were analyzed using the DYNAMICS software package (Protein Solutions Inc.) and were averaged.

2.2. Crystallization and X-ray data collection

Initial crystallization attempts were carried out at 293 and 278 K using a 96-condition crystallization screen originally designed by the Mitsubishi Chemical Corporation with the sitting-drop vapour-diffusion method. Crystals were observed under many crystallization conditions and a crystallization condition containing 100 mM MES–NaOH buffer pH 6.5, 15%(w/v) PEG 8000 and 200 mM potassium thiocyanate was further optimized. Finally, diffraction-quality crystals were obtained at 293 K in 100 mM sodium citrate buffer pH 5.6 containing 16%(w/v) PEG 8000, 150 mM potassium thiocyanate using the sitting-drop vapour-diffusion method (Fig. 2 [triangle]). The optimized crystallization drops were prepared by mixing 0.5 µl protein solution and an equal volume of reservoir solution and were equilibrated against 100 µl reservoir solution. The crystals appeared reproducibly within 3 d and the largest crystal grew to dimensions of approximately 0.8 × 0.03 × 0.03 mm (Fig. 2 [triangle]).

Figure 2
Crystals of BAS grown by the sitting-drop method.

A single crystal was divided into two halves of length 0.4 mm with a Micro-Scraper (Hampton Research) in order to fit into a 0.4 mm nylon loop. The crystal was transferred to reservoir solution containing 20%(v/v) glycerol as a cryoprotectant. After a few seconds, the crystal was picked up again in a nylon loop and flash-cooled at 100 K in a nitrogen-gas stream. X-ray diffraction data sets were collected on SPring-8 beamline BL24XU using a Rigaku R-­AXIS V imaging-plate area detector. The wavelength of the synchrotron radiation was 0.82656 Å and the distance between the crystal and the detector was 350 mm. A total of 180 frames were recorded with 1° oscillation and 48 s exposure time. Data were indexed, integrated and scaled with the HKL-2000 program package (Otwinowski & Minor, 1997 [triangle]).

3. Results and discussion

The recombinant BAS was heterologously expressed in E. coli as a fusion protein with GST at the N-terminus. After cleavage of the GST tag, the purified enzyme migrated as a single band with a molecular weight of 43 kDa on SDS–PAGE, which agrees well with the calculated value of 42.4 kDa. In contrast, a gel-filtration experiment yielded a molecular weight of 83 kDa, suggesting that the recombinant BAS is a homodimeric enzyme, as in the case of other known type III PKSs (Austin & Noel, 2003 [triangle]). The DLS analysis after gel filtration revealed a monomodal distribution, with a polydispersity value of 14.0% and an estimated molecular weight of 81 kDa, which are in good agreement with the results of the gel-filtration analysis.

A complete data set was collected to 1.8 Å resolution. From the diffraction data collection, the space group was determined to be P21, with unit-cell parameters a = 54.6, b = 89.6, c = 81.1 Å, α = γ = 90.0, β = 100.5°. Detailed data-processing statistics are shown in Table 1 [triangle]. With two monomers in the asymmetric unit, the Matthews volume (V M; Matthews, 1968 [triangle]) was calculated to be 2.3 Å3 Da−1 and the estimated solvent content was 43.7%, which is in the range normally observed for protein crystals. Further structure determination using the molecular-replacement method, with the crystal structure of the M. sativa CHS (PDB code 1bq6; Ferrer et al., 1999 [triangle]) as a search model, is in progress.

Table 1
Data-collection statistics


This work was supported in part by a grant from the National Project on Protein Structural and Functional Analyses.


  • Abe, I., Oguro, S., Utsumi, Y., Sano, Y. & Noguchi, H. (2005). J. Am. Chem. Soc.127, 12709–12716. [PubMed]
  • Abe, I., Sano, Y., Takahashi, Y. & Noguchi, H. (2003). J. Biol. Chem.278, 25218–25226. [PubMed]
  • Abe, I., Takahashi, Y., Morita, H. & Noguchi, H. (2001). Eur. J. Biochem.268, 3354–3359. [PubMed]
  • Abe, T., Morita, H., Noma, H., Kohno, T., Noguchi, H. & Abe, I. (2007). Bioorg. Med. Chem. Lett.17, 3161–3166. [PubMed]
  • Austin, M. B., Bowman, M. E., Ferrer, J.-L., Schroder, J. & Noel, J. P. (2004). Chem. Biol.11, 1179–1194. [PubMed]
  • Austin, M. B. & Noel, J. P. (2003). Nat. Prod. Rep.20, 79–110. [PubMed]
  • Ferrer, J.-L., Jez, J. M., Bowman, M. E., Dixon, R. A. & Noel, J. P. (1999). Nature Struct. Biol.6, 775–784. [PubMed]
  • Jez, J. M., Austin, M. B., Ferrer, J., Bowman, M. E., Schroder, J. & Noel, J. P. (2000). Chem. Biol.7, 919–930. [PubMed]
  • Jez, J. M., Ferrer, J.-L., Bowman, M. E., Dixon, R. A. & Noel, J. P. (2000). Biochemistry, 39, 890–902. [PubMed]
  • Matthews, B. W. (1968). J. Mol. Biol.33, 491–497. [PubMed]
  • Morita, H., Kondo, S., Oguro, S., Noguchi, H., Sugio, S., Abe, I. & Kohno, T. (2007). Chem. Biol.14, 359–369. [PubMed]
  • Otwinowski, Z. & Minor, W. (1997). Methods Enzymol.276, 307–326.

Articles from Acta Crystallographica Section F: Structural Biology and Crystallization Communications are provided here courtesy of International Union of Crystallography