Poster: Secondary metabolism

Abs # 251: Molecular characterization of tetrahydrocannabinolic acid synthase and cannabidiolic acid synthase from Cannabis sativa

Presenter:	Sirikantaramas, Supaart , supaarts@yahoo.com
Authors	Sirikantaramas, Supaart  (A)   Taura, Futoshi  (A)   Morimoto, Satoshi  (A)   Shoyama, Yukihiro  (A)
Affiliations:	(A): Graduate School of Pharmaceutical Sciences, Kyushu University, Japan

Delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are well-known cannabinoids found only in Cannabis sativa. THC and CBD are derived from tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA), respectively. Previously we have reported that THCA and CBDA are biosynthesized from cannabigerolic acid (CBGA). In this report, we discuss cloning and characterization of two biosynthetic enzymes, tetrahydrocannabinolic acid synthase (THCAS) and cannabidiolic acid synthase (CBDAS), to understand their reaction mechanisms. The genes encoding THCAS and CBDAS were cloned by PCR. Interestingly, deduced amino acid sequence of THCAS and CBDAS share 82% identity. Both enzymes are cloned and expressed in insect cells using baculovirus expression system. Molecular weights of THCAS and CBDAS have been reduced by PNGase treatment, suggesting that they are modified by N-linked glycosylation. THCAS shows same biochemical properties as those of THCAS from C. sativa. Further characterization showed that THCAS is a member of flavoprotein family structurally related oxidoreductases. THCAS contains a covalently linked FAD cofactor with a molar ratio of FAD to protein at 1:1. Site-directed mutagenesis suggested that the site of covalent attachment is His-114. Moreover, we have constructed a transgenic THCA-producing tobacco hairy root which can produce THCA by exogenous addition of biosynthetic precursor, CBGA. CBDAS contains a covalently linked FAD cofactor. However, CBDAS activity is very low. From SDS-PAGE analysis, deglycosylated CBDAS has molecular weight of 54 kDa, which is lower than the theoretical molecular weight, 59 kDa. This result suggests that CBDAS undergoes proteolysis. Further investigations to determine the cleavage site are now in progress.