Crystallization of Delta1-tetrahydrocannabinolic acid (THCA) synthase from Cannabis sativa

Abstract

Delta1-Tetrahydrocannabinolic acid (THCA) synthase is a novel oxidoreductase that catalyzes the biosynthesis of the psychoactive compound THCA in Cannabis sativa (Mexican strain). In order to investigate the structure-function relationship of THCA synthase, this enzyme was overproduced in insect cells, purified and finally crystallized in 0.1 M HEPES buffer pH 7.5 containing 1.4 M sodium citrate. A single crystal suitable for X-ray diffraction measurement was obtained in 0.09 M HEPES buffer pH 7.5 containing 1.26 M sodium citrate. The crystal diffracted to 2.7 A resolution at beamline BL41XU, SPring-8. The crystal belonged to the primitive cubic space group P432, with unit-cell parameters a = b = c = 178.2 A. The calculated Matthews coefficient was approximately 4.1 or 2.0 A3 Da(-1) assuming the presence of one or two molecules of THCA synthase in the asymmetric unit, respectively.

Figure 1

THCA biosynthesis. THCA synthase catalyzes oxidative cyclization of the monoterpene moiety of CBGA to form THCA. THC is derived from THCA by non-enzymatic decarboxylation.

Figure 2

Crystals of THCA synthase. The scale bar is 0.1 mm in length.