Photon Factory Activity Report 2017 #35 (2018)

BL-1A/2016G507

X-Ray Crystal Structure Analysis of Plant Type III Polyketide Synthase
Chin Piow WONG1 and Hiroyuki MORITA1,*1
Institute of Natural Medicine, University of Toyama, Toyama 930-0194, Japan
1 Introduction
Type III polyketide synthases (PKSs) perform C-C
bond forming reactions by iterative Claisen-typecondensation of CoA thioesters and cyclization of thepoly-β-keto intermediates, to produce pharmaceuticallyand biologically important aromatic polyketides, such as
chalcone, stilbene, and curcumin. Tetraketide synthase
PoOLS (Primula obconca olivetol synthase) is a plantderived type III PKS involved in the biosynthesis ofprimin. PoOLS is proposed to catalyze the condensationsof one hexanoyl-CoA and three malonyl-CoA to generate
a linear pentyltetraketide-CoA, which is cyclized byanother enzyme into olivetolic acid and further modified
into the final product primin (Figure 1). Apart from its
native substrate, PoOLS is able to accept CoA substrates
with sidechain up to 11 carbons as starter unit. In effort tounderstand substrate and product specificities, we proceed
to determine the X-ray crystal structure of PoOLS.

using the Dali Program [1] indicated that PoOLS is most
homologically related to OsPKS, a type III PKS identified
from Oryza sativa, with Cα RMSD at 0.9 Å. PoOLS
catalytic cavity is tunnel-shaped, resembling the catalytic
cavity of PKS18, a mycobacterial type III PKS [2]. The
obtained result suggests that PoOLS favors C6-C12
aliphatic-CoAs as a starter substrate to produce linear C5C11 fatty acyltetraketide-CoAs. However, further
structure-based site-directed mutagenesis studies ofPoOLS will provide insight into the catalytic versatility ofthe type III PKSs.

SCoAOO
hexanoyl-CoASCoA
C5H11OO+HOSCoAOOHOCOOH
C5H11O
H3COC5H11O

OHO

pentyltetraketide-CoA

olivetolic acidpriminmalonyl-CoA

Fig. 1: The proposed biosynthetic pathway of primin.
2 Materials and Methods
Crystallization – Diffraction-quality crystals of PoOLS
were obtained after few days of incubation at 4 ºC, in 100
mM Tris-HCl (pH 8.8) containing 20% PEG2000, with22 mg/mL of purified PoOLS solution, by using sittingdrop vapor-diffusion method.
Data collection – The crystals were transferred into thesoaking solution with 5% (v/v) glycerol for 10 sec forcryoprotection and then flash cooled at -173°C in anitrogen-gas stream. The X-ray diffractions of crystals
were collected at BL1A, processed and scaled with XDS.
The structure was solved by the molecular replacement
method with Phaser-MR (simple one-componentinterface) using alkylquinolone synthase (AQS, PDB
accession code: 5wx4) as search model. The structure wasmodified manually with Coot and refined with PHENIX.
3 Results and DiscussionThe crystal structure of PoOLS was solved at 1.8 Å.
The final R-value was 20.6% (Rfree = 26.6%). PoOLS
formed a symmetric dimer and showed the typical type III
pilyketide synthase fold. Significant difference between
the overall structures of PoOLS and the other type III
PKSs were not observed (Figure 2). Homology search

Fig. 2: Structure of the PoOLS. Lauric acid and glycerol
are colored green and blue sticks, respectively.
Acknowledgement
We sincerely thank the PF staffs for their help with ourdata collections.
References[1] L. Holm, C. Sander, Trends Biochem. Sci. 20, 478480 (1995).
[2] R. Sankaranarayanan, P. Saxena, U.B. Marathe, R.S.
Gokhale, V.M. Shanmugam, R. Rukmini, Nat. Struct.
Mol. Biol. 11, 894-900 (2004)* hmorita@inm.u-toyama.ac.jp