Photon Factory Activity Report 2018 #36 (2019)

BL-1A/2017G011

X-Ray Crystal Structure Analysis of Plant Type III Polyketide Synthase
Chin Piow WONG , Takahiro MORI , Takayoshi AWAKAWA , Ikuro ABE , and Hiroyuki MORITA
Institute of Natural Medicine, University of Toyama, Toyama 930-0194, Japan
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan
12221,*121 Introduction
Type III polyketide synthase (PKS) catalyzes theiterative Claisen-type condensation of the CoA thioestersand cyclization of the poly-β-keto intermediates, toproduce pharmaceutically and biologically class ofcompounds, such as chalcone, stilbene, and curcumin.
Recently, a type III PKS is implicated to be also involvedin the biosynthesis of tropane alkaloids [1], which is aclass of compounds that include the pharmaceuticallyimportant hyoscyamine and scopolamine. The AbPYKS
identified from Atropha belladonna was revealed to playa role in the biosynthesis of tropinone, a key tropane
intermediate. It is proposed that AbPYKS catalyzes a
non-canonical reaction of N-methyl-Δ1-pyrrolium cation
with two round of elongation with malonyl-CoA
mediated elongation to yield 4-(1-methyl-2-pyrrolidinyl)3-oxobutanoic acid, which is further tailored intotropinone by a cytochrome P450 reductase (Figure 1).
However, the detailed enzymatic mechanism employed
by AbPYKS remains unknown. Hence, we crystallized
the AbPYKS and study its 3-dimensional structure inorder to reveal its catalytic mechanism.
OAbPYKSNN

N-methyl-Δ1-pyrroliumO
2 x HOO

P450

4-(1-Methyl-2-pyrrolidinyl)-3-oxobutanoic acidONOHO
TropinoneSCoAFig. 1: Proposed biosynthetic pathway for the formationof tropinone catalyzed by AbPYKS.
2 Materials and Methods
Crystallization – Diffraction-quality crystals of AbPYKS
were obtained after one week of incubation at 20 ºC, in
100 mM HEPES (pH 7.5) containing 20% PEG4000 with
15 mg/mL of purified AbPYKS solution, by using sittingdrop vapor-diffusion method.
Data collection – The crystals were transferred into thesoaking solution with 20% (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 curcuminoid synthase (msCHS, PDB ID:1BI5) as model. The structure was modified manuallywith Coot and refined with PHENIX.

Fig. 2: Overall structure of the AbPYKS. The catalytic
triad, Cys167, His306, and Asn339 is highlighted in green.
3 Results and Discussion
The crystal structure of AbPYKS was solved by Xray crystallography at 2.7 Å resolution. The final R-value
was 17.9% (Rfree = 25.4%). AbPYKS adopts the typicalhomodimeric construct and ababa-fold architecture that
commonly occurs in other type III polyketide synthases(Figure 2). Structural homology search using the DaliProgram [2] indicated that AbPYKS is most structurally
related to stilbene synthase identified from Vitis vinifera,with Ca RMSD at 0.8 Å (PDB ID: 3TSY). Comparisonsof the molecular structure of AbPYKS with other type III
PKSs and further investigation of the AbPYKS crystal
structure complexed with its substrates could highlightthe important residues and catalytic mechanism the latteremployed to catalyze its non-canonical mechanism.
Acknowledgement
We sincerely thank the PF staffs for their help with ourdata collections.
Reference[1] M.A. Bedewitz, A.D. Jones, J.C. D’Auria, C.S. Barry,
Nat Comm. 9 5281 (2018)[2] L. Holm, C. Sander, Trends Biochem. Sci. 20, 478480 (1995).
* hmorita@inm.u-toyama.ac.jp