and modulation were confirmed by site directed mutational analysis (Shoyama et al.
2012 ). For example, Tyr484 was found to most likely act as the general base in the
reaction to abstract the proton from the phenolic hydroxyl group of CBGA. His292
appears to take part in the reaction as a counter ion residue and interact with the
carboxyl group of the substrate. In addition, Tyr417, which is not essential for
catalysis, might stabilize the substrate binding via hydrogen bonding. Recent
computational substrate docking simulations also supported these deduced func-
tions for the respective amino acids in the active site (Alaoui et al. 2014 ).
The reaction mechanism of THCA synthase, based on biochemical and structural
studies, is summarized in Fig.8.5. In this mechanism, the reaction is initiated by a
hydride transfer from the C-1 position of CBGA to the reactive N-5 position of the
FAD isoalloxazine ring. Meanwhile, a proton is removed from the hydroxyl group
of the substrate to form an ionic intermediate. In thefinal step of the reaction, a
stereoselective ring closure forms THCA, which takes place in the active site of
THCA synthase. The hydride ion is then transferred from the reducedflavin to
molecular oxygen, resulting in hydrogen peroxide formation and re-activation of
theflavin for the next reaction cycle.
8.2.1.2 Localization and Possible Physiological Function
It was reported that THC accumulates only in the secretory cavity of the glandular
trichomes ofC. sativa(Fairbairn 1972 ; Kim and Mahlberg 1997 ). Thus, we sus-
pected that THCA is also biosynthesized in the glandular trichomes. To investigate
Fig. 8.5 The reaction mechanism of tetrahydrocannabinolic acid (THCA) synthase. R represents
the remainder of the FAD molecule. The substrate and product are indicated in red. Amino acid
residues involved in substrate binding and catalysis are indicated in blue. CBGA, cannabigerolic
acid
8 Cannabinoids: Biosynthesis and Biotechnological Applications 189