# Biosynthesis of Nature-Inspired Unnatural Cannabinoidss
## 4 Cannabinoid Synthases  
The enzymatic reaction catalyzed by the cannabinoid synthases is probably one of the most challenging steps in the cannabinoid biosynthetic pathway. These synthase enzymes, such as the more well-studied THCA synthase and CBDA synthase, are FAD (flavin adenine dinucleotide)-dependent oxidoreductase that are not easy to express heterologously in bacterial systems. The need for post-translational modifications, such as glycosylation and vacuolar compartmentalization for functional expression and protein folding, meant that eukaryotic hosts are arguably the better option [19]. Out of the three cannabinoid synthases identified (THCA synthase, CBDA synthase, CBCA synthase), THCA synthase is the most well-studied as it is the only cannabinoid synthase from _C. sativa_ that is crystallized [70] and solved structurally [45]. This is followed by CBDA synthase, which has been isolated, purified and heterologously expressed in yeast and insect cells [27; 71]. CBCA synthase has been extracted and purified from young leaves of _C. sativa_ and partially characterized in 1998 [72; 73]. However, no further in-depth research or reports of heterologous expression has been conducted since. In 2012, [PERSON] et al. solved the crystal structure of THCA synthase, allowing us to understand the reaction mechanism from a structural perspective [45]. The oxidative cyclization of CBGA to form THCA was presumed to proceed via two parts: a chemical bond between C1\({}^{\prime}\) and C6\({}^{\prime}\) of the geranyl moiety of CBGA via elimination of the hydride on C1\({}^{\prime}\), and a second chemical bond between the O atom of the hydroxyl group on C4 of CBGA and C7\({}^{\prime}\) on the geranyl moiety through a proton extraction from the hydroxyl group. The FAD moiety was ascertained to be absolutely critical for catalysis and responsible for the hydride transfer on C1\({}^{\prime}\) via the N5 atom of the isoalloxazine ring in FAD, analogous to the reaction mechanism in homologues from berberine bridge enzymes (BBE). Through structural alignments with BBE homologues and further mutational analysis, they concluded Tyr484 is the basic residue in THCA synthase responsible for the remaining deprotonation of the O atom on C4 of CBGA and proposed a reaction mechanism, as depicted in Figure 10.  
The high level of sequence similarity (\(\sim\)83%) between the THCA synthase and CBDA synthase indicates that they share a common ancestor [27]. More specifically, it was recently suggested that THCA synthase evolved from CBDA synthase through a duplication and divergence event, giving rise to a different product profile producing dominantly THCA instead of CBDA [74]. Using partially purified protein samples of THCA and CBDA syntheses, [PERSON] et al. studied their respective product profiles when using CBGA as substrate  
Figure 10: Proposed reaction mechanism of THCA synthase enzyme. The substrate and product are colored black; amino acid residues from THCA synthase are colored blue and the FAD moiety is colored red. and found that both enzymes produced the same products, albeit at different ratios, in a pH-dependent manner [71]. They subsequently tried to investigate how differences in amino acid residues in the active sites of both enzymes translate to differences in the product profile. First, an attempt was made to reconstruct a CBDA-dominant product profile using a THCA synthase scaffold by mutating the different amino acid residues in the THCA synthase active site to that of CBDA synthase. Unfortunately, this abolished all catalytic activity which led them to further pursue single amino acid changes of THCA synthase residues to that of CBDA synthase, and vice versa, in a bid to identify the residues responsible for the different cyclization mechanisms.