The Gene Controlling Marijuana Psychoactivity

Abstract

⌬ 1 -Tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes oxidative cyclization of cannabigerolic acid into THCA, the precursor of ⌬ 1 -tetrahydrocannabinol. We cloned a novel cDNA (GenBank TM accession number AB057805) encoding THCA synthase by reverse transcription and polymerase chain reactions from rapidly expanding leaves of Cannabis sativa. This gene consists of a 1635-nucleotide open reading frame, encoding a 545-amino acid polypeptide of which the first 28 amino acid residues constitute the signal peptide. The predicted molecular weight of the 517-amino acid mature polypeptide is 58,597 Da. Interestingly, the deduced amino acid sequence exhibited high homology to berberine bridge enzyme from Eschscholtzia californica, which is involved in alkaloid biosynthesis. The liquid culture of transgenic tobacco hairy roots harboring the cDNA produced THCA upon feeding of cannabigerolic acid, demonstrating unequivocally that this gene encodes an active THCA synthase. Overexpression of the recombinant THCA synthase was achieved using a baculovirus-insect expression system. The purified recombinant enzyme contained covalently attached FAD cofactor at a molar ratio of FAD to protein of 1:1. The mutant enzyme constructed by changing His-114 of the wild-type enzyme to Ala-114 exhibited neither absorption characteristics of flavoproteins nor THCA synthase activity. Thus, we concluded that the FAD binding residue is His-114 and that the THCA synthase reaction is FAD-dependent. This is the first report on molecular characterization of an enzyme specific to cannabinoid biosynthesis.

Benzylisoquinoline alkaloid biosynthesis in opium poppy

“…In BIA metabolism, FADOXs have only been implicated in the formation of C-C and C-N bonds, the most noteworthy being the berberine bridge enzyme (BBe), which generates the protoberberine scaffold (Dittrich and Kutchan 1991;Facchini et al 1996;winkler et al 2006). In contrast, FADOX homologs appear to only catalyze the formation of C-O bonds in the formation of non-alkaloid specialized metabolites in other plants (Custers et al 2004;Sirikantaramas et al 2004). Although ODDs are known to perform a variety of hydroxylations, epoxidations and desaturations in a variety of plant metabolic pathways (Prescott and John 1996), enzymes from this family have only been shown to catalyze the dealkylation of certain BIA subgroups (Hagel and Facchini 2010;Farrow and Facchini 2013).…”

A concerted mechanism for berberine bridge enzyme

“…These new mechanistic ideas are potentially also relevant for two closely related enzymes: Δ 1 -tetrahydrocannabinolic acid (THCA) synthase and cannabidiolic acid (CBDA) synthase from Cannabis sativa 18,19 . Both enzymes also catalyze an oxidative C-C coupling reaction, which is proposed to proceed via a two-step mechanism, where ring closure is an ionic process following the oxidation of the substrate 18,19 .…”

“…Both enzymes also catalyze an oxidative C-C coupling reaction, which is proposed to proceed via a two-step mechanism, where ring closure is an ionic process following the oxidation of the substrate 18,19 . Due to the similarity of the catalyzed reactions and the significant sequence similarity to BBE (~40% identity), it is conceivable that these two enzymes operate by an analogous one-step concerted mechanism.…”

6-S-Cysteinylation of Bi-covalently Attached FAD in Berberine Bridge Enzyme Tunes the Redox Potential for Optimal Activity

“…⌬ 1 -tetrahydrocannabinolic acid synthase from Cannabis sativa (8). Most of these enzymes with a confirmed or proposed bi-covalently attached flavin have a function in specialized metabolic pathways and catalyze reactions with rather challenging chemistry or substrates.…”