420 is officially brought to you by THCA Synthase!! Here’s how…
THCA synthase is the enzyme that catalyzes the reaction of CBGA to THCA, creating the THC precursor known to display psychoactive properties in C. sativa. The structure of THCA synthase was determined using X-ray crystallography. THCA and CBDA synthases share 83% similarity, with CBDA synthase proposed to be the ancestral synthase from which the THCA variant evolved. As CBDA synthase is the ancestral enzyme, THCA synthase has retained the ability to form CBDA as a product as well. In fact THCA synthase can make THCA, CBDA, and CBCA in a pH dependent manner. CBDA is a minor side product while THCA is the dominant product in pH below 7 while this changes to CBCA being the dominant product in pH above 7. The change is believed to be the result in the different protonation step at H292 (pH 6.04).
The reaction that THCA synthase carries out is a cyclization reaction that is carried out through an oxidation reaction and is assisted by the FAD cofactor. The reaction results in the formation of two 6 membered rings fused together. The alcohol group is converted to a cyclic ester in the formation of the heterocyclic ring. One of the double bonds in CBGA is lost in the formation of the fused ring structure for THCA while the other is moved in position.
Deprotonation of CBGA occurs via the phenolate ion of Y484, kicking off the cyclization cascade. This is followed by hydride transfer at C3 to the FAD cofactor, oxidizing the substrate. Hydride transfer is C3 of CBGA to N5 of FAD, followed by deprotonation at O6’ of CBGA. Catalysis does not involve E442 which is the catalytic base in other BBE, but THCA synthase loses all enzymatic activity without the hydroxyl of Y484.
In addition to being held by the covalent bonds at H114 and C176, the FAD cofactor is also held in place by hydrogen bonds to backbone nitrogen atoms at Gly113, His114, Tyr175, Gly180, and Gly183, as well as two side chain nitrogen atoms on His184 and Asn483, and also two side chain hydroxyl groups on Tyr190 and Tyr481. No mention of water molecules playing a role in the active site was reported.
Part of the p-cresol methyl-hydroxylase superfamily of enzymes. THCA synthase has a tertiary structure that contains two domains on one chain, with the FAD coenzyme located between the domains bound covalently to H114 and C176, which are both part of domain I. In both THCA and CBDA synthases, the H114A substitution results in loss of enzymatic activity. Domain I is from amino acid residues 28-253 and 476-545 with 8 alpha helices and 8 beta sheets, while domain II is residues 254-475 and has 8 antiparallel beta sheets surrounding 6 alpha helices.
THCA synthase has a C-terminal domain known as the berberine-bridge-enzyme (BBE) domain and as such THCA synthase is also part of the BBE-like family of enzymes. The BBE spans from amino acid residues 477 to 535. While the BBE lacks any secondary structures, the BBE does contain the important Y484 residue. Other highly conserved amino acid residues in the BBE region include Y510, F531 and Q535, all of which displayed no enzymatic activity when replaced with an alanine variant.
In yeast models altered for the production of THCA synthase, the knockout of one or two N-glycosylation sites resulted in increased enzymatic activity while knockout of numerous sites led to decreased activity with knockout of all seven glycosylation sites resulting in no enzymatic activity at all. These results indicate that the N-glycosylations may have more to do with enzyme expression than with catalytic activity.
References:
- Zirpel, Bastian, Oliver Kayser, and Felix Stehle. “Elucidation of structure-function relationship of THCA and CBDA synthase from Cannabis sativa L.” Journal of biotechnology 284 (2018): 17-26.
- Shoyama, Yoshinari, et al. “Structure and function of∆ 1-tetrahydrocannabinolic acid (THCA) synthase, the enzyme controlling the psychoactivity of Cannabis sativa.” Journal of molecular biology 423.1 (2012): 96-105.