# Biosynthesis of Nature-Inspired Unnatural Cannabinoidss
## 4 Cannabinoid Synthases
### Protein Engineering  
Cannabinoid synthases such as THCA and CBDA synthases are interesting targets of engineering, as these enzymes use the same substrate (CBGA) but produce different products, depending on how they cyclize the C10 geranyl moiety in CBGA. As mentioned prior, the THCA synthase reaction mechanism starts with Tyr484 extracting the proton on O6\({}^{\prime}\), which initiates the reaction cascade that leads to the formation of two chemical bonds. To form a CBDA product in comparison, a chemical bond is formed between C1\({}^{\prime}\) and C6\({}^{\prime}\) of the geranyl moiety in CBGA and an electron transfer from C8 to C9. CBDA synthase is postulated to obtain the proton from C8 of the geranyl group, which subsequently initiates the reaction cascade leading to a CBDA product (Figure 11). A sequence alignment between THCA synthase and CBDA synthase, without the plastid targeting signal in the first 28 residues, shows that there are 85 amino acid differences. Of these 85 amino acid residue differences (\(\sim\)16.4%), 44 amino acids (\(\sim\)8.5%) are amino acid substitutions with strongly similar properties, 17 amino acids (\(\sim\)3.3%) are substitutions with weakly similar properties, while 24 residues (\(\sim\)4.6%) are non-conserved. A rational protein engineering project aiming to investigate the reaction mechanism pertaining to product specificity of these cannabinoid synthases would be to look at these amino acid differences, starting from the non-conserved residues to the most conserved residues. However, the matrix might still be too large for any efficient screen. [PERSON] et al. attempted to narrow down the sequence space by using a targeted approach of looking at amino acid differencesclose to the THCA synthase active site, but did not manage to find any particular residue or set of residues that are key to this differential product specificity [71]. A closer inspection of the difference in reaction mechanism between THCA and CBDA synthase in Figure 11 reveals the following: (1) both reactions proceed through an FAD-assisted hydride transfer at C1\({}^{\prime}\) of the geranyl moiety on CBGA and an additional base-facilitated dehydration; (2) the two reactions diverge at the position of the proton extraction by the basic residue (proposed to be Tyr484 in THCA synthase and Tyr483 in CBDA synthase, respectively); (3) in CBDA synthase, Tyr483 extracts the proton from the terminal methyl group of the geranyl moiety [27], instead of the O atom of the hydroxyl group on C4 of CBGA in THCA synthase. Taken together, the residues involved in the reaction mechanism are highly similar. However, the architecture of the active site in CBDA synthase, surrounding the geranyl moiety of CBGA, would possibly be different, causing the C10 carbon chain to orientate itself differently, and hence, resulting in the extraction of proton from the end of the chain, instead of the O atom of the C4 hydroxyl group, as seen in THCA synthase. Using THCA synthase as the template, we created homology models of CBDA synthase and CBCA synthase and compared the structures using a structural alignment (Figure 12). Figure 12 highlights the different amino acid residues found in the active site of the three cannabinoid synthases, analogous to the work of [PERSON] et al. However, instead of single amino acid changes, a subset of amino acids could be investigated, such as Val415 and Thr446, changing to the corresponding Ala414 and Ile445, respectively, in CBDA synthase, as these residues are found on the lateral side closer to the prenyl group on the docked CBGA substrate. A sequential step with an increasing number of residues changed could follow until a set of key residues responsible for causing the prenyl group to orientate in a different manner, leading to the difference in cyclization, is found. Interestingly, [PERSON] et al. also investigated the role of the carboxylate functional group in CBGA in catalysis.