THCA synthase catalyzes a unique monoterpene cyclase-like reaction coupled
with a two-electron oxidation. However, the deduced primary structure of THCA
synthase was not similar to that of the monoterpene cyclases that cyclize geranyl
pyrophosphate (Croteau 1987 ). THCA synthase had unexpectedly high homology
to the berberine bridge enzyme, a vanillyl alcohol oxidase (VAO) familyflavin
adenine dinucleotide (FAD) oxidase, involved in alkaloid biosynthesis (Dittrich and
Kutchan 1991 ). The VAOflavoprotein family includes various plant enzymes
associated with secondary metabolism (Leferinka et al. 2008 ; Dijkman et al. 2013 ),
among them, THCA synthase is thefirst that catalyzes terpenophenol biosynthesis.
High levels of expression of the recombinant enzyme by a baculovirus insect
expression system promoted biochemical studies on this novel cannabinoid syn-
thase. These studies indicated that the enzyme reaction is a typical FAD oxidase
type as reported for berberine bridge enzyme (Kutchan and Dittrich 1995 ); the
reaction is dependent on the FAD coenzyme and molecular oxygen, and releases
THCA and hydrogen peroxide in a 1:1 molar ratio. On the other hand, the bio-
chemical approaches were not informative concerning the active site structure and
functions of respective amino acid residues.
Therefore, Shoyama et al. ( 2012 ) prepared crystals of the recombinant THCA
synthase (Fig.8.4), and unequivocally determined the tertiary structure of THCA
synthase by X-ray crystallographic analysis at a resolution of 2.75Å. The most
typical feature of THCA synthase was covalent attachment to the FAD coenzyme
via 6-S-cysteinyl, 8a-N1-histidyl linkages at His114 and Cys176. This novel
bi-covalent linkage to FAD has also been identified for homologous plant enzymes,
including the berberine bridge enzyme fromEschscholzia californicaand mono-
lignol oxidase (AtBBE-like15) fromArabidopsis thaliana, of which crystallo-
graphic studies have already been reported (Winkler et al. 2008 ; Daniel et al. 2015 ).
Based on the active site architecture of THCA synthase, the amino acid residues
important for the reaction were identified, and their functions in substrate binding
Fig. 8.4 Crystals of the
recombinant
tetrahydrocannabinolic acid
(THCA) synthase. The
crystals are yellow due to
flavin adenine dinucleotide
(FAD)-binding. The bar
represents 0.1 mm.
Reproduced with permission
from Taura et al. (2009a).
Copyright Wiley-VHCA,
Zürich, Switzerland
188 S. Sirikantaramas and F. Taura