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We identified a unique enzyme that catalyzes the oxidocyclization of cannabigerolic acid to cannabidiolic acid (CBDA) in Cannabis sativa L. (CBDA strain). The enzyme, named CBDA synthase, was purified to apparent homogeneity by a four-step procedure: ammonium sulfate precipitation followed by chromatography on DEAE-cellulose, phenyl-Sepharose CL-4B, and hydroxylapatite. The active enzyme consists of a single polypeptide with a molecular mass of 74 kDa and a pI of 6.1. The NH2-terminal amino acid sequence of CBDA synthase is similar to that of Δ1-tetrahydrocannabinolic-acid synthase. CBDA synthase does not require coenzymes, molecular oxygen, hydrogen peroxide, and metal ion cofactors for the oxidocyclization reaction. These results indicate that CBDA synthase is neither an oxygenase nor a peroxidase and that the enzymatic cyclization does not proceed via oxygenated intermediates. CBDA synthase catalyzes the formation of CBDA from cannabinerolic acid as well as cannabigerolic acid, although the kcat for the former (0.03 s−1) is lower than that for the latter (0.19 s−1). Therefore, we conclude that CBDA is predominantly biosynthesized from cannabigerolic acid rather than cannabinerolic acid.
Cannabinoids are plant secondary metabolites possessing alkylresorcinol (typically olivetol or olivetolic acid) and monoterpene groups in their molecules (Fig. 1). Numerous cannabinoids have been isolated from marijuana or fresh Cannabis leaves, and their chemical and pharmacological properties have been extensively investigated (
), while cannabidiol reduces aggressive behavior in the L-pyroglutamate-treated rat, spontaneous dyskinesias in the dystonic rat, and turning behavior in the 6-hydroxyldopamine-treated rat caused by apomorphine (
Several pathways have been proposed to explain the biosynthesis of these cannabinoids. Typical biosynthetic schemes have been based on the assumptions that Δ1-tetrahydrocannabinolic acid (Δ1-THCA), the precursor of Δ1-tetrahydrocannabinol, is synthesized by the ring closure of CBDA and that CBDA is formed from cannabigerolic acid (CBGA) via hydroxyl-CBGA (
). To confirm these assumptions, we investigated Δ1-THCA biosynthesis by enzymological means and established that Δ1-THCA is actually biosynthesized from CBGA by Δ1-THCA synthase and not from the presumed precursor, CBDA (
). In contrast, it is still unknown whether the biosynthesis of CBDA proceeds through the above biosynthetic pathway. This lack of a precise understanding of CBDA biosynthesis is mostly due to the fact that the enzymes involved in CBDA formation have not hitherto been studied.
To understand the mechanism of CBDA biosynthesis, we investigated the enzymes involved in the production of CBDA. Consequently, we identified a unique enzyme (named CBDA synthase) that catalyzes the stereoselective oxidocyclization of CBGA to CBDA in the rapidly expanding leaves of the CBDA strain. In this paper, we describe the purification and biochemical properties of CBDA synthase. In addition, we present evidence that CBDA is biosynthesized from CBGA through oxidocyclization without hydroxylation.
Despite a lack of experimental evidence, it has been believed that CBDA is biosynthesized from CBGA via hydroxyl-CBGA (
). To definitively establish the biosynthetic mechanism of CBDA, we directly investigated the enzyme (CBDA synthase) that catalyzes the formation of CBDA. Since this enzyme had not been studied, we first attempted to identify CBDA synthase under various conditions. After unsuccessful attempts, CBDA synthase could be extracted with phosphate buffer from the CBDA strain. Higher enzyme activity was observed in rapidly expanding leaves than in mature leaves. This distribution of CBDA synthase correlates well with the CBDA content; a higher amount of CBDA is found in rapidly expanding leaves (11.9 mg/g of fresh leaves) than in mature leaves (3.3 mg/g of fresh leaves).2 These findings indicate that CBDA is predominantly biosynthesized by CBDA synthase in rapidly expanding leaves of the CBDA strain. Previously, we demonstrated that biosynthesis of Δ1-THCA also predominantly occurs in rapidly expanding leaves of the Mexican strain (
). The roles of CBDA and Δ1-THCA in plants remain largely unclear, but these cannabinoids may play an important role in leaf development.
CBDA synthase was purified 519-fold by a four-step procedure that yielded up to 15% final recovery of the enzyme activity. Purification of this enzyme to homogeneity permitted the characterization of its precise properties, resulting in a variety of new findings. In particular, it is noteworthy that the oxidocyclization of CBGA by CBDA synthase is not accompanied by oxygenation (Fig. 4), contrary to the published hypothesis of CBDA biogenesis. Oxygenase-type enzymes catalyzing the cyclization of terpene groups have been identified in several plants (
) have demonstrated that deguelin, the isoflavonoids in Tephrosia vogelii, is formed through the prenyl cyclization of rot-2-enoic acid by deguelin cyclase and that, like CBDA formation, this reaction proceeds through direct hydrogenation without a cofactor requirement. However, it is quite difficult to precisely compare the kinetic and physical properties of deguelin cyclase and CBDA synthase since deguelin cyclase was not purified to homogeneity.
Concerning the substrate specificity, CBDA synthase catalyzes the formation of CBDA from CBNRA as well as CBGA (Fig. 4). Since the C-1-C-2 double bond of CBDA has the same configuration as that of CBNRA, CBDA formation could proceed from CBGA through CBNRA to CBDA. However, CBDA synthase displayed much higher activity for CBGA than CBNRA, indicating that CBNRA is not an intermediate in the oxidocyclization of CBGA into CBDA. In addition, the lower substrate specificity for CBNRA suggests that CBDA is biosynthesized predominantly from CBGA rather than CBNRA. This is supported by the fact that the content of CBNRA in the CBDA strain is much lower than that of CBGA (0.08 versus 2.8 mg/g of rapidly expanding leaves).2
Kinetic properties similar to those of CBDA synthase have been described for limonene synthase, which mediates the formation of limonene with higher Vmax and lower Km values for geranyl pyrophosphate as compared with neryl pyrophosphate (
) has proposed that these metal ions might neutralize the negative charge of the diphosphate moiety and assist in ionization of the allylic diphosphate substrate. Since CBGA has no allylic diphosphate moiety, it is reasonable that CBDA synthase has no requirement for Mg2+ and Mn2+. Although the low turnover number for CBGA (0.19 s−1) suggests that CBDA synthase also may require some cofactors, we could not demonstrate either cofactors or coenzymes that activate the enzyme activity. However, since a much lower or a similar turnover number (kcat = 0.01-0.3 s−1) has been reported for some terpene cyclases (
), Δ1-THCA synthase catalyzes the oxidocyclization of CBGA with a higher turnover number (0.20 s−1) for CBGA than for CBNRA, and this reaction has no requirement for cofactors, coenzymes, and molecular oxygen. In addition, the molecular mass, pI, and NH2-terminal sequence of both enzymes are quite similar. Although CBDA has a different ring system from Δ1-THCA, these similarities suggest that both cannabinoids are formed by a similar reaction mechanism.
We thank Yoshitsugu Tanaka, Kyoko Soeda, and Dr. Ryuichi Isobe for NMR and mass measurements of cannabinoids. We acknowledge Dr. Yuji Ito for helpful advice and discussions.