The gene controlling marijuana psychoactivity: molecular cloning and heterologous expression of Delta1-tetrahydrocannabinolic acid synthase from Cannabis sativa L.
Delta(1)-tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes oxidative cyclization of cannabigerolic acid into THCA, the precursor of Delta(1)-tetrahydrocannabinol. We cloned a novel cDNA (GenBank trade mark 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.
Sirikantaramas
Supaart
S
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, 812-8582, Japan.
Morimoto
Satoshi
S
Shoyama
Yoshinari
Y
Ishikawa
Yu
Y
Wada
Yoshiko
Y
Shoyama
Yukihiro
Y
Taura
Futoshi
F
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002188:Cannabis / Q000737:chemistry / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary; D013759:Dronabinol / Q000096:biosynthesis* / Q000737:chemistry; D007313:Insecta; D019746:Intramolecular Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D018517:Plant Roots / Q000502:physiology; D014026:Nicotiana; D014162:Transfection
D018076:DNA, Complementary; D013759:Dronabinol; D019746:Intramolecular Oxidoreductases; C100677:delta(1)-tetrahydrocannabinolic acid synthase