Photon Factory Activity Report 2019 #37 (2020)

BL-1A/2018G522

X-Ray Crystal Structure Analysis of Plant Type III Polyketide Synthase,PECPS
Chin Piow WONG1, Qian Qian Liu1, Shepo SHI2, and Hiroyuki MORITA1,*

1Institute of Natural Medicine, University of Toyama, Toyama 930-0194, Japan

2Modern Research Center for Traditional Chinese Medicine, School of Chinese Materia

Medica, University of Chinese Medicine, Beijing, 100029, China

1 Introduction
Type III polyketide synthases (PKSs) catalyze theiterative Claisen-type condensation of the CoA
thioesters and cyclization of the poly-β-ketointermediates, to produce pharmaceutically andbiologicallyimportantcompounds.
Phenylethylchromone precursor synthase (PECPS)
from agarwood, Aquilaria sinensis, is a type III PKS
that is thought to be involved in the biosynthesis of 2(2-phenylethyl)chromones(PECs),
whichisimportant aromatic ingredient in perfumery, oriental
medicines, incensing ceremony, and crafts
production [1]. Thus, to unveil the intimate catalytic
mechanism of PECPS would provide insight into not
only the catalytic diversity of type III PKSs, but also
engineering of the enzyme to generate PEC analogs.
Hence, we solved the crystal structure of PECPS.
2 Experiment
Crystallization – Diffraction-quality crystals ofPEPCS were obtained at 20 ºC, in 100 mM Tris-HCl(pH 8.5) containing 0.12 M KF, 4% butanediol, and
24% PEG8000 with 15 mg/mL of purified PECPS
solution, by using sitting-drop vapor-diffusion
method.
Data collection – The crystals were transferred into
the soaking solution with 20% (v/v) glycerol for 10 sec
for cryoprotection and then flash cooled at -173°C ina nitrogen-gas stream. The X-ray diffractions ofcrystals were collected at BL1A, processed andscaled with XDS. The structure was solved by themolecular replacement method with Phaser-MR(simple one-component interface) using OsPKS(PDB entry 4YJY) as model. The structure wasmodified manually with Coot and refined withPHENIX.
3 Results and Discussion
The crystal structure of PECPS was solved by Xray crystallography at 1.95 Å resolution. The final Rvalue was 19.9% (Rfree = 23.7%). PECPS adopts thetypical homodimeric construct and αβαβα-foldarchitecture that commonly occurs in other type III
polyketide synthases (Fig. 1). Analyses of the cavity
size using the CASTp Program [2] indicated that thecatalytic cavity of PECPS (247 Å3) is significantly
smaller than OsCUS (642 Å3), a type III PKS thatcatalyzes
theone-pot
formationofbisdemethoxycurcumin from the condensation of two
p-coumaroyl-CoA and one malonyl-CoA [3]. Further
analyses of amino acid residues lining the catalytic
cavity of PECPS in conjunction with activity-basedsite-directed mutagenesis might provide further
insight into the catalytic mechanism of PECPS.

Fig. 1: Overall structure of the PECPS. The catalytic
triad, Cys166, His308, and Asn338, is highlighted ingreen. Met139 protruding into adjoining monomer arehighlighted in magenta.
Acknowledgement
We sincerely thank the PF staffs for their help with
our data collections.
References[1] X. H. Wang, B. W. Gao, X. Liu, X. J. Dong, Z. X.
Zhang, H. Y. Fan, L. Zhang, J. Wang, S. P. Shi, P.
F. Tu, BMC Plant Biol. 2016, 16, 119.
[2] W. Tian, C. Chen, X. Lei, J. Zhao, J. Lian, Nucleic
Acid Res. 2018, 46, 363-367.
[3] H. Morita, K. Wanibuchi, H. Nii, R. Kato, S. Sugio,I. Abe, Proc. Natl. Acad. Sci. USA 2010, 107,19778-19783.
* hmorita@inm.u-toyama.ac.jp