Photon Factory Activity Report 2016 #34 (2017)

BL-17A, AR-NE3A, AR-NW12A/2015G007

Crystal structure analysis of plant polyketide cyclcase OAC from Cannabis sativa

Takashi Matsui1, Xinmei Yang1, Xiaoxi Zhou1, Futoshi Taura2, Hiroshi Noguchi3, Ikuro Abe4 andHiroyuki Morita1,*
1
Institute of Natural Medicine, University of Toyama, Toyama, 930-194, Japan
2
Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, 9300194, Japan3
School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, 422-8526, Japan
4
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, 113-0033, Japan
1 Introduction
Polyketides are a structurally diverse family of naturalproducts. In the biosynthesis of plant polyketides, theconstruction of the ring formation is a key step indiversifying the polyketide structures. Olivetolic acidcyclase (OAC) derived from Cannabis sativa L., involvedin cannabinoid biosynthesis, is the only known plant
polyketide cyclase. OAC catalyzes the C2-C7
intramolecular aldol cyclization of linear pentyl tetra-bketide CoA to generate olivetolic acid (OA). To clarify
the intimate catalytic mechanism of OAC, structures ofseveral OAC mutants were determined.
2 Experiment
Crystallization – 20 mg/ml OAC I7F, Y27W, V59M,
Y72F an H78S mutants were crystallized with sittingdrop vapor diffusion method, respectively. 1 µl of protein
and 1 µl of reservoir solution were mixed, andequilibrated against 50 µl of reservoir solution at 5°C.
Diffraction-quality mutants crystals were finally obtained
after a few days incubation.
Data collection and structure determination – Crystals
were transferred into the reservoir solution containing
10%(v/v) glycerol as a cryoprotectant, and were flashfrozen in a nitrogen stream. The X-ray diffraction data ofmutant crystals were collected at beamline BL-17A,NE3A, and NW-12A under cryogenic condition at -173°C,respectively. The diffreaction data was processed andscaled usnig XDS. The structures of OAC mutants weresolved by the molecular replacement method with Molrepor Phaser using OAC apo as the search model. The
structures of OAC apo and OAC-OA complex modified
mannually with Coot and refined with phenix.refine. The
coordinates and structure factors have been depositedunder accession number 5B0B for the I7F, 5B0D for theY27W, 5B0E for the V59M, 5B0F for the Y72F and5B0G for the H78S mutant, respectively.
3 Results and Discussion
X-ray structures of OAC I7F, Y27W, V59M, Y72F, andH78S were refined at 2.18, 1.80, 1.60, 1.60, and 1.40 Åresolutions, respectively. The final R-values of I7F, Y27W,
V59M, Y72F, and H78S mutant structures were 20.1%,19.6%, 21.2%, 17.5%, and 17.4% (Rfree = 25.4%, 24.5%,23.5%, 22.3% and 20.0%), respectively. The structuralcomparisons couplied with enzymatic reaction revealed

that the Y72F and H78S, which lost the catalytic activity,substitutions only disrupt the hydrogen bond bwetween
Tyr72 and His78 at the active site with no significant
conformational change. Furthermore, The I7F, Y27W, andV59M mutants, which located on the pentyl-binding
pocket and showed 15%, 63% and 35% decreases in OAforming activity, exhibited estimated total cavity volumesof 247, 204, and 222 Å3, which are slightly less than the270 Å3 volume of the wild type. These observations
strongly favor the possible catalytic roles of Tyr72 andHis78 over their roles in substrate binding affinity, andthe crucial roles of the pentyl-binding pocket in thebinding affinites of the pentyl moiety of the substrate,
respectively.

Fig. 1: The active-site architectures of wild-type OAC
and the OAC mutant enzymes. The active-sitearchitectures of (A) OAC and the OAC mutant enzymes(B) H78S, (C) Y72F, (D) Y27W, (D) I7F, and (H) V59M.
The OA molecule bound in the wild-type OAC structure
is superimposed on the active-site cavity of the mutant
enzymes, and depicted by a green stick model. The vander Waals force of OA and the hydrogen bonds are
depicted by dotted surfaces and light blue dashed lines,
respectively.
References[1] X. Yang et al., Acta Cryst. F71, 1470-1474 (2015).
[2] X. Yang et al, FEBS J. 283, 1088-1106 (2016).
* hmorita@inm.u-toyama.ac.jp