transfer step: CBDA synthase abstracts a proton from the terminal methyl group of
CBGA, whereas a proton is removed from the hydroxyl group of the substrate in
the THCA synthase reaction. After the proton elimination, stereoselective ring
closure takes place in the active sites of each respective cannabinoid synthase,
forming CBDA and THCA.
Due to structural and functional similarities, only a small number of amino acid
residues likely determine the product specificity of these cannabinoid synthases.
The structural basis differentiating these reactions remains unclear, as the tertiary
structure of CBDA synthase is not available. Further studies, including crystal
structure analysis of CBDA synthase, are needed to reveal the differences between
the two cannabinoid synthases for the rational design of the biotechnological cat-
alysts, for example, producing a specific blend of THCA and CBDA for medicinal
purposes, as in the case of Sativex (Syed et al. 2014 ).
8.2.2.2 Cannabidiolic Acid Synthase, the Chemotype-Determining
Enzyme
CBDA synthase and THCA synthase are also of interest from a genetic point of view,
as these enzymes determine the well-known drug-type (marijuana) andfiber-type
(hemp) chemotypes ofC. sativa. Some research groups have attempted to crossbreed
the two chemotypes, and consistently obtained similar results (Yotoriyama et al.
1980 ; de Meijer et al. 2003 ). When pure drug andfiber chemotypes were crossed, all
of the F 1 plants were mixed chemotypes that produced both THCA and CBDA. In
addition, subsequent inbreeding of the F 1 plants resulted in the production of three F 2
chemotypes (drug-type, mixed chemotype, andfiber-type) in a 1:2:1 segregation
pattern. Based on the codominant inheritance of the two chemotypes, it was believed
that THCA synthase and CBDA synthase were encoded by two alleles at the same
locus in theCannabisplant genome (de Meijer et al. 2003 ).
On the other hand, another explanation proposing that two enzymes are encoded
by two linked yet genetically separate loci, has also been suggested (van Bakel et al.
2011 ). More recently, Weiblen et al. ( 2015 ) provided evidence supporting that
THCA synthase and CBDA synthase are encoded in two separate regions of the
Cannabisgenome, based on the sequence diversity of these cannabinoid synthases,
the position of enzyme coding loci on the genome, and the pattern of gene
expression. Thus, gene duplication and divergence are the most probable reasons
explaining why THCA synthase and CBDA synthase, the chemotype-determining
enzymes, have evolved to synthesize different cannabinoids.
8.2.3 Cannabichromenic Acid Synthase
CBCA is a minor constituent produced by both drug-type andfiber-type (Shoyama
et al. 1975 )C. sativaplants. It is also known that this cannabinoid is most actively
192 S. Sirikantaramas and F. Taura