CBDA synthase
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CBDA synthase (cannabidiolic acid synthase, CBDAS) is the enzyme that turns cannabigerolic acid (CBGA) into cannabidiolic acid (CBDA), the compound from which cannabidiol (CBD) forms when the acid loses carbon dioxide. It is the dominant cannabinoid enzyme of the fibre and grain crop.[1] With THCA synthase and CBCA synthase it forms the set of cannabinoid oxidocyclases, all three drawing on the same precursor.[2]
Futoshi Taura, Satoshi Morimoto and Yukihiro Shoyama purified the enzyme in 1996 from the rapidly expanding leaves of a CBDA-rich plant, and the gene was cloned in 2007, the second of the pathway to be isolated.[3][1] Whether a plant carries it in working form, and whether it also carries a working THCA synthase, settles the plant's chemotype.[4]
The reaction
The enzyme closes a ring in the geranyl part of CBGA to give CBDA, an oxidative cyclisation of the same kind that THCA synthase performs on the same molecule. The two differ at one step: CBDA synthase takes a proton from a terminal methyl group of the substrate where THCA synthase takes one from a hydroxyl group, and that choice decides which ring forms.[1][5]
The 1996 study of the purified enzyme found no requirement for a coenzyme, for molecular oxygen or for a metal ion, and concluded that the cyclisation does not run through an oxygenated intermediate.[3] Work on the recombinant protein in 2007 revised that: like THCA synthase it is a covalently flavinylated oxidase that uses oxygen and releases hydrogen peroxide.[1]
The enzyme also accepts cannabinerolic acid, the cis isomer of CBGA, but converts it about six times more slowly, so CBDA in the plant comes mainly from CBGA.[3] Its products are not confined to CBDA. Recombinant CBDA synthase supplied with CBGA also yields small amounts of THCA and cannabichromenic acid (CBCA).[6] Measured at pH 5 and 30 °C it was the most selective of the cannabinoid oxidocyclases tested, giving 89 per cent CBDA, 8 per cent CBCA and 3 per cent THCA, and also the least active, at an eighth of the activity of THCA synthase.[7]
Protein and structure
The purified enzyme is a single polypeptide with a measured mass of 74 kilodaltons and an isoelectric point of 6.1.[3] The coding sequence gives 517 amino acids and a calculated mass of about 59 kilodaltons; the difference is attributed to sugars added to the protein after it is made.[5] Its sequence is 83.9 per cent identical to that of THCA synthase, and the two were proposed to descend from a common ancestor.[1]
The structure was solved in 2026, at 1.70 ångström the sharpest of the three cannabinoid synthases, with the flavin bound in the active site. The three share their fold, and CBDA synthase is the one that stands apart: 84 per cent of its sequence matches THCA synthase and 82 per cent cannabichromenic acid synthase. Tyrosine 483, tryptophan 443, tyrosine 190 and histidine 291 are conserved across all three, while substitutions around the active site of CBDA synthase change its shape and the tunnel through which the substrate reaches it, which suits the different ring it closes. A serine missing from CBDA synthase accounts for the offset of one in residue numbering between it and its relatives.[8]
Replacing serine 116 with alanine, or alanine 414 with valine, raised the activity for CBDA about threefold; the second change also gave the enzyme a nineteenfold higher activity for THCA and widened the range of pH over which it worked.[6]
The gene and its forms
The gene has no intron and a full-length coding copy is found in CBDA-producing plants.[2] It was cloned from a fibre-crop plant, and two further copies in the same plant's DNA made no active enzyme.[1]
Functional sequences fall into two types. One is found in fibre varieties such as Ermo, in the cannabidiol-rich drug cultivars CBDRx and Jamaican Lion, and in landraces from China and Japan; the other in the fibre varieties Finola, Carmen, Ermes, Futura 75, Tygra and Uso 31. Alongside the fully working forms are weakened and defective ones, including a variant from Turkey whose plants accumulate part of their cannabinoid as CBGA, and variants from hybrid cultivars of Italy and Ukraine in which CBGA predominates.[2][9]
Plants of the drug crop carry sequences 93 per cent identical to the working gene which share a deletion of four bases and so encode a shortened protein. These sequences are strictly associated with plants that make THCA, and up to three of them have been found in a single cultivar.[2] The deletion separated drug-crop from fibre-crop plants in a panel of 21 varieties.[10] Their close linkage to THCA synthase is why they serve as markers for the drug crop, and why an assay that tells the working gene from the shortened one predicts how much THCA and CBDA a plant will make.[2]
Genotype and the ratio
Feral plants of Minnesota were genotyped at the gene and measured for cannabinoids. The three chemotype groups, CBD-type, intermediate and THC-type, occurred at frequencies of 0.88, 0.11 and 0.01, and matched the genotypes without exception: plants homozygous for the non-working form had THC-type ratios, homozygotes for the working form CBD-type ratios and heterozygotes intermediate ones. A test cross segregated as codominant Mendelian inheritance predicts. The genotype also predicted the ratio in 20 clinical cultivars supplied blind, and in Canadian grain cultivars wherever plants held more than 0.5 per cent total cannabinoids.[4]
The total amount of cannabinoid a plant makes is a separate matter from the ratio, and varied widely among plants of the same genotype.[4] In a cross between a drug-type cultivar and the hemp Carmen, the one locus for the ratio accounted for 92 per cent of the variation in the second generation, while variation in total content was associated with other chromosomes.[11]
Position in the genome
A map made from a cross between the drug strain Purple Kush and the hemp variety Finola placed the gene at a locus close to, but separate from, that of THCA synthase. Finola carries one working copy of CBDA synthase and no THCA synthase; Purple Kush carries THCA synthase and a disabled copy of CBDA synthase, broken by an inserted gypsy element. Each gene lies inside a stretch of more than 250 kilobases rich in retrotransposons which has almost nothing in common with its counterpart in the other parent, and both stretches lie within about 40 megabases of repetitive DNA where crossing over is rare.[12]
The copies sit in tandem arrays inside long terminal repeat retrotransposons on chromosome 7. The cannabidiol-rich cultivar CBDRx is mostly of drug-crop ancestry and carries its working CBDA synthase on a stretch of hemp ancestry, with no complete THCA synthase; only CBDA synthase was expressed in cannabidiol-type plants, while plants of intermediate ratio expressed both genes.[11] Across 193 assembled genomes no haploid genome held more than one full-length copy of either gene, and the full-length copies lay among cassettes of transposable elements that mostly hold pseudogenes, within about 1.5 megabases on chromosome 7. The genes themselves carried very little variation although the surrounding region is among the most variable in the species.[13]
Where the enzyme came from
Because sequences resembling CBDA synthase vary more than those resembling THCA synthase, CBDA synthase was taken for the ancestral form of the family.[9] A phylogeny built from whole genomes put that variety down to further gene lineages that had been read as variants of one gene, and placed the working CBDA synthase sequences in a clade of their own.[2] Ancestral enzymes reconstructed from 77 sequences of cannabis, hop and related plants and made in yeast were unselective, and the oldest active one made more THCA than anything else, so neither living specialist stands for the ancestral state; the specialists arose after duplication, by divergence.[7]
Making the enzyme outside the plant
The enzyme has been produced in the yeast Komagataella phaffii, the route by which the protein for the structural work was obtained.[6][8] A strain of Saccharomyces cerevisiae carrying the whole pathway, CBDA synthase included, made CBDA from galactose alone, and the same work made the propyl cannabinoids tetrahydrocannabivarinic acid and cannabidivarinic acid.[14]
See also
References
- ↑ 1.0 1.1 1.2 1.3 1.4 1.5 Taura, F.; Sirikantaramas, S.; Shoyama, Y.; Yoshikai, K.; Shoyama, Y.; Morimoto, S. (2007). "Cannabidiolic-acid synthase, the chemotype-determining enzyme in the fiber-type Cannabis sativa". FEBS Letters. 581 (16): 2929–2934. doi:10.1016/j.febslet.2007.05.043.
- ↑ 2.0 2.1 2.2 2.3 2.4 2.5 van Velzen, R.; Schranz, M.E. (2021). "Origin and evolution of the cannabinoid oxidocyclase gene family". Genome Biology and Evolution. 13 (8) evab130. doi:10.1093/gbe/evab130.
- ↑ 3.0 3.1 3.2 3.3 Taura, F.; Morimoto, S.; Shoyama, Y. (1996). "Purification and characterization of cannabidiolic-acid synthase from Cannabis sativa L.: biochemical analysis of a novel enzyme that catalyzes the oxidocyclization of cannabigerolic acid to cannabidiolic acid". Journal of Biological Chemistry. 271 (29): 17411–17416. doi:10.1074/jbc.271.29.17411.
- ↑ 4.0 4.1 4.2 Wenger, J.P.; Dabney, C.J.; ElSohly, M.A.; Chandra, S.; Radwan, M.M.; Majumdar, C.G.; Weiblen, G.D. (2020). "Validating a predictive model of cannabinoid inheritance with feral, clinical, and industrial Cannabis sativa". American Journal of Botany. 107 (10): 1423–1432. doi:10.1002/ajb2.1550. PMC 7702092.
- ↑ 5.0 5.1 Tahir, M.N.; Raz, F.S.; Rondeau-Gagné, S.; Trant, J.F. (2021). "The biosynthesis of the cannabinoids". Journal of Cannabis Research. 3 (1) 7. doi:10.1186/s42238-021-00062-4.
- ↑ 6.0 6.1 6.2 Zirpel, B.; Kayser, O.; Stehle, F. (2018). "Elucidation of structure-function relationship of THCA and CBDA synthase from Cannabis sativa L.". Journal of Biotechnology. 284: 17–26. doi:10.1016/j.jbiotec.2018.07.031.
- ↑ 7.0 7.1 Villard, C.; Baser, I.; van de Peppel, A.C.; Cankar, K.; Schranz, M.E.; van Velzen, R. (2025). "Resurrected ancestral cannabis enzymes unveil the origin and functional evolution of cannabinoid synthases". Plant Biotechnology Journal. 24 (4): 2685–2697. doi:10.1111/pbi.70475.
- ↑ 8.0 8.1 Domenech, J.; King, A.; Byrne, E.; Cartwright, J.; Grogan, G. (2026). "X-ray crystal structures of the cannabinoid synthases CBCAS, CBDAS and THCAS". Current Research in Structural Biology. 12 100197. doi:10.1016/j.crstbi.2026.100197.
- ↑ 9.0 9.1 Onofri, C.; de Meijer, E.P.M.; Mandolino, G. (2015). "Sequence heterogeneity of cannabidiolic- and tetrahydrocannabinolic acid-synthase in Cannabis sativa L. and its relationship with chemical phenotype". Phytochemistry. 116: 57–68. doi:10.1016/j.phytochem.2015.03.006.
- ↑ Cascini, F.; Farcomeni, A.; Migliorini, D.; Baldassarri, L.; Boschi, I.; et al. (2019). "Highly predictive genetic markers distinguish drug-type from fiber-type Cannabis sativa L." Plants. 8 (11) 496. doi:10.3390/plants8110496.
- ↑ 11.0 11.1 Grassa, C.J.; Weiblen, G.D.; Wenger, J.P.; Dabney, C.; et al. (2021). "A new Cannabis genome assembly associates elevated cannabidiol (CBD) with hemp introgressed into marijuana". New Phytologist. 230 (4): 1665–1679. doi:10.1111/nph.17243. PMC 8248131.
- ↑ Laverty, K.U.; Stout, J.M.; Sullivan, M.J.; Shah, H.; Gill, N.; Holbrook, L.; Deikus, G.; Sebra, R.; Hughes, T.R.; Page, J.E.; van Bakel, H. (2019). "A physical and genetic map of Cannabis sativa identifies extensive rearrangements at the THC/CBD acid synthase loci". Genome Research. 29 (1): 146–156. doi:10.1101/gr.242594.118. PMC 6314170.
- ↑ Lynch, R.C.; Padgitt-Cobb, L.K.; Garfinkel, A.R.; Knaus, B.J.; Hartwick, N.T.; et al. (2025). "Domesticated cannabinoid synthases amid a wild mosaic cannabis pangenome". Nature. 643 (8073): 1001–1010. doi:10.1038/s41586-025-09065-0. PMC 12286863.
- ↑ Luo, X.; Reiter, M.A.; d'Espaux, L.; Wong, J.; Denby, C.M.; et al. (2019). "Complete biosynthesis of cannabinoids and their unnatural analogues in yeast". Nature. 567 (7746): 123–126. doi:10.1038/s41586-019-0978-9. PMC 6830570.