# Biosynthesis of Phytocannabinoids and Structural Insights: A Review
## 5 Structural Analysis of the Enzymes THCA and CBDA Synthase  
In this study, an in silico approach was used to perform structural analysis (1D, 2D, and 3D) of enzymes such as THC acid and CBD acid synthase. The primary compositions of the nucleotide and protein sequences were predicted using BioEdit V 7.2.6.1 [51] software, as represented in Supplementary Table S1. The pairwise sequence alignment of THC acid and CBD acid synthase was performed using the Clustal Omega server ([[https://www.ebi.ac.uk/Tools/msa/clustalo/](https://www.ebi.ac.uk/Tools/msa/clustalo/)]([https://www.ebi.ac.uk/Tools/msa/clustalo/](https://www.ebi.ac.uk/Tools/msa/clustalo/)) accessed on 15 February 2023). The alignment shows 90.6% of sequences are similar to one another, as depicted in Figure 3. Whereas the secondary structural elements of target enzymes were predicted using the (SOPMA) server ([[https://npsa-prabi.ibcp.fr/NPSA/npsa_sopma.html/](https://npsa-prabi.ibcp.fr/NPSA/npsa_sopma.html/)]([https://npsa-prabi.ibcp.fr/NPSA/npsa_sopma.html/](https://npsa-prabi.ibcp.fr/NPSA/npsa_sopma.html/)) accessed on 15 February 2023). The results revealed that a maximum number of residues formed a random coil, which determines the stability of enzymes. Further, 170 amino acid residues were found to be involved in alpha-helix and 132 for extended strand formation, as shown in Figures 4a and 5a.  
Protein 3D structures were modeled with the Swiss Model [52], and the predicted structure was confirmed with the Ramachandran Plot server ([[https://zlab.umassmed.edu/bu/rama/accessed](https://zlab.umassmed.edu/bu/rama/accessed)]([https://zlab.umassmed.edu/bu/rama/accessed](https://zlab.umassmed.edu/bu/rama/accessed)) on 15 February 2023). The predicted model exhibited 100% similarity with the THCA enzymes and 83.72% for CBDAs, using the protein data bank (PDB) template 3 VTE, as depicted in Figures 4b and 5b, respectively. According to the structural validation results (Ramachandran plot), 441 and 438 amino acid residues were found in the allowed region, which occupies 97.351% and 96.689% for THCA and CBDA synthase, as represented in Figures 4c and 5c, respectively. Our target enzyme's motif and domain region were predicted using the smart server and Conserved Domain Database NCBI-CDD [53] search. The prediction revealed that two major domains, the FAD binding domain at positions 81-219 and Berberine bridge-like domain at positions 480-538, were found in both THCA and CBDA enzymes (Figures 4d and 5d, respectively). Interestingly, a low-complexity region (repeats of single amino acids) was found in position 6-27 bases  
Figure 4: Prediction of the 1D, 2D, and 3D structural profile of Tetrahydrocannabinolic acid synthase. (**a**) Secondary structural elements, (**b**) Predicted 3D model, (**c**) Validated model, and (**d**) Predicted motif and domain regions.  
only in CBD [54]. In the 1960s and 1970s, numerous plausible hypotheses were advanced regarding the biosynthesis of THCA; however, they all were lacking experimental support. THCA was thought to arise from CBDA through cyclization. However, the reaction conditions of the transformation do differ from those present during natural biosynthesis in the plants; moreover, isomerase activity, which would be necessarily responsible for the conversion of CBDA into THCA, has never been detected in any enzyme assays using crude _C. sativa_ enzyme extracts. Current thinking suggests it comes from CBGA instead, by either tetrahydroocannabinolic acid synthase (THCAs) or cannabidiolic acid synthase (CBDAs), both members of the p-cesol methyl-hydroxylase super family.