Tetrahydrocannabinolic acid (THCA) synthase (full name ''Δ¹-tetrahydrocannabinolic acid synthase'') is an

enzyme Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products ...

responsible for catalyzing the formation of THCA from

cannabigerolic acid Cannabigerolic acid (CBGA) is the acidic form of cannabigerol (CBG). It is a dihydroxybenzoic acid and olivetolic acid in which the hydrogen at position 3 is substituted by a geranyl group. It is a biosynthetic precursor to Delta-9-tetrahydrocann ...

(CBGA). THCA is the direct

precursor Precursor or Precursors may refer to: * Precursor (religion), a forerunner, predecessor ** The Precursor, John the Baptist Science and technology * Precursor (bird), a hypothesized genus of fossil birds that was composed of fossilized parts of u ...

of tetrahydrocannabinol (THC), the principal psychoactive component of

cannabis ''Cannabis'' () is a genus of flowering plants in the family Cannabaceae. The number of species within the genus is disputed. Three species may be recognized: '' Cannabis sativa'', '' C. indica'', and '' C. ruderalis''. Alternative ...

, which is produced from various strains of ''

Cannabis sativa ''Cannabis sativa'' is an annual herbaceous flowering plant indigenous to Eastern Asia, but now of cosmopolitan distribution due to widespread cultivation. It has been cultivated throughout recorded history, used as a source of industrial fibe ...

''. Therefore, THCA synthase is considered to be a key enzyme controlling cannabis psychoactivity. Polymorphisms of THCA synthase result in varying levels of THC in Cannabis plants, resulting in "drug-type" and "fiber-type" ''C. sativa'' varieties.

Structure

THCA synthase is a 60

kDa The dalton or unified atomic mass unit (symbols: Da or u) is a non-SI unit of mass widely used in physics and chemistry. It is defined as of the mass of an unbound neutral atom of carbon-12 in its nuclear and electronic ground state and at re ...

(~500 amino acids) monomeric enzyme with the

isoelectric point The isoelectric point (pI, pH(I), IEP), is the pH at which a molecule carries no net electrical charge or is electrically neutral in the statistical mean. The standard nomenclature to represent the isoelectric point is pH(I). However, pI is also u ...

at 6.4.

Post-translational Post-translational modification (PTM) is the covalent and generally enzymatic modification of proteins following protein biosynthesis. This process occurs in the endoplasmic reticulum and the golgi apparatus. Proteins are synthesized by ribosom ...

N-linked glycosylation ''N''-linked glycosylation, is the attachment of an oligosaccharide, a carbohydrate consisting of several sugar molecules, sometimes also referred to as glycan, to a nitrogen atom (the amide nitrogen of an asparagine (Asn) residue of a protein), i ...

increases the total mass to approximately 74 kDa. The

tertiary structure Protein tertiary structure is the three dimensional shape of a protein. The tertiary structure will have a single polypeptide chain "backbone" with one or more protein secondary structures, the protein domains. Amino acid side chains may i ...

is divided into two domains (domains I and II), with a flavin adenine dinucleotide (FAD) positioned between the two domains. Domain I comprises eight

alpha helices The alpha helix (α-helix) is a common motif in the secondary structure of proteins and is a right hand-helix conformation in which every backbone N−H group hydrogen bonds to the backbone C=O group of the amino acid located four residues ear ...

and eight beta sheet and is covalently bound to FAD. Domain II comprises five alpha helices surrounded by eight beta sheets. Enzymes that share similar amino acid sequences include the

flavoprotein Flavoproteins are proteins that contain a nucleic acid derivative of riboflavin. Flavoproteins are involved in a wide array of biological processes, including removal of radicals contributing to oxidative stress, photosynthesis, and DNA repair. T ...

s berberine bridge enzyme (BBE), glucooligosaccharide oxidase (GOOX), and aclacinomycin oxidoreductase (AknOx). The FAD moiety is the location of enzymatic activity and is covalently bound to His114 and Cys176. FAD is also bound by hydrogen bonds with neighboring amino acid main chains and side chains. The co-crystallization of THCA synthase with substrate or product has not been accomplished yet. File:FAD in THCA synthase.png, FAD cofactor (in green) is located between domain I and domain II of THCA synthase. Alpha helices are cyan and beta sheets are magenta. File:FAD active site.png, FAD (in green) is covalently bound to histidine 114 and cysteine 176 (in pink).

Reaction mechanism

THCA synthase, a

, uses a flavin adenine dinucleotide (FAD) cofactor to

catalyze Catalysis () is the process of increasing the rate of a chemical reaction by adding a substance known as a catalyst (). Catalysts are not consumed in the reaction and remain unchanged after it. If the reaction is rapid and the catalyst recyc ...

the

oxidative Redox (reduction–oxidation, , ) is a type of chemical reaction in which the oxidation states of substrate change. Oxidation is the loss of electrons or an increase in the oxidation state, while reduction is the gain of electrons or a d ...

cyclization A cyclic compound (or ring compound) is a term for a compound in the field of chemistry in which one or more series of atoms in the compound is connected to form a ring. Rings may vary in size from three to many atoms, and include examples where ...

of the

monoterpene Monoterpenes are a class of terpenes that consist of two isoprene units and have the molecular formula C10H16. Monoterpenes may be linear (acyclic) or contain rings (monocyclic and bicyclic). Modified terpenes, such as those containing oxygen func ...

moiety Moiety may refer to: Chemistry * Moiety (chemistry), a part or functional group of a molecule ** Moiety conservation, conservation of a subgroup in a chemical species Anthropology * Moiety (kinship), either of two groups into which a society is ...

of cannabigerolic acid (CBGA). Similar cyclization reactions occur in monoterpene biosynthesis from

geranyl pyrophosphate Geranyl pyrophosphate (GPP), also known as geranyl diphosphate (GDP), is the pyrophosphate ester of the terpenoid geraniol. Its salts are colorless. It is a precursor to many natural products. Occurrence GPP is an intermediate in the isopreno ...

, but are not oxidative. THCA synthase exhibits no catalytic activity against

cannabigerol Cannabigerol (CBG) is one of more than 120 identified cannabinoid compounds found in the plant genus ''Cannabis''. Cannabigerol is the decarboxylated form of cannabigerolic acid, the parent molecule from which other cannabinoids are synthesized ...

, which lacks a

carboxyl In organic chemistry, a carboxylic acid is an organic acid that contains a carboxyl group () attached to an R-group. The general formula of a carboxylic acid is or , with R referring to the alkyl, alkenyl, aryl, or other group. Carboxylic ...

group compared to CBGA, suggesting that the carboxyl group of CBGA is necessary for the reaction to occur. The overall chemical reaction is: CBGA + O₂

\rightleftharpoons

THCA + H₂O₂ Tetrahydrocannabinolicacid

A hydride is transferred from CBGA to reduce FAD, concerted by

deprotonation Deprotonation (or dehydronation) is the removal (transfer) of a proton (or hydron, or hydrogen cation), (H+) from a Brønsted–Lowry acid in an acid–base reaction.Henry Jakubowski, Biochemistry Online Chapter 2A3, https://employees.csbsju. ...

of a

hydroxyl In chemistry, a hydroxy or hydroxyl group is a functional group with the chemical formula and composed of one oxygen atom covalently bonded to one hydrogen atom. In organic chemistry, alcohols and carboxylic acids contain one or more hydro ...

group by a tyrosine residue. The monoterpene moiety in CBGA is then positioned to complete cyclization into THCA. Oxidation of reduced FAD by O₂ produces hydrogen peroxide (H₂O₂). THCA synthase mechanism

Biological function

THCA synthase is expressed in the glandular

trichome Trichomes (); ) are fine outgrowths or appendages on plants, algae, lichens, and certain protists. They are of diverse structure and function. Examples are hairs, glandular hairs, scales, and papillae. A covering of any kind of hair on a p ...

s of ''

''. THCA synthase may contribute to the

self-defense Self-defense (self-defence primarily in Commonwealth English) is a countermeasure that involves defending the health and well-being of oneself from harm. The use of the right of self-defense as a legal justification for the use of force ...

of ''Cannabis'' plants by producing THCA and

hydrogen peroxide Hydrogen peroxide is a chemical compound with the formula . In its pure form, it is a very pale blue liquid that is slightly more viscous than water. It is used as an oxidizer, bleaching agent, and antiseptic, usually as a dilute solution (3%� ...

, which are both cytotoxic. Because these products are toxic to the plant, THCA synthase is secreted into the trichome storage cavity. THCA also acts as a necrosis-inducing factor by opening

mitochondrial permeability transition pore The mitochondrial permeability transition pore (mPTP or MPTP; also referred to as PTP, mTP or MTP) is a protein that is formed in the inner membrane of the mitochondria under certain pathological conditions such as traumatic brain injury and stro ...

s, inhibiting mitochondrial viability and resulting in senescence in leaf tissues. Non-enzymatic decarboxylation of THCA during storage or smoking forms

THC Tetrahydrocannabinol (THC) is the principal psychoactive constituent of cannabis and one of at least 113 total cannabinoids identified on the plant. Although the chemical formula for THC (C21H30O2) describes multiple isomers, the term ''THC' ...

, the principal psychoactive component of cannabis. Further degradation by temperature, auto-oxidation, and light forms cannabinol. THC and other

cannabinoid Cannabinoids () are several structural classes of compounds found in the cannabis plant primarily and most animal organisms (although insects lack such receptors) or as synthetic compounds. The most notable cannabinoid is the phytocannabinoid tet ...

s are well-known to reduce nausea and vomiting and stimulate hunger, particularly in patients undergoing

cancer chemotherapy Chemotherapy (often abbreviated to chemo and sometimes CTX or CTx) is a type of cancer treatment that uses one or more anti-cancer drugs ( chemotherapeutic agents or alkylating agents) as part of a standardized chemotherapy regimen. Chemothera ...

. Similar enzymes to THCA synthase catalyze the formation of other cannabinoids. For example, cannabidiolic acid (CBDA) synthase is a flavoprotein that catalyzes a similar oxidative cyclization of CPGA into CBDA, the dominant cannabinoid constituent of fiber-type ''C. sativa''. CBDA undergoes a similar decarboxylation to form

cannabidiol Cannabidiol (CBD) is a phytocannabinoid discovered in 1940. It is one of 113 identified cannabinoids in cannabis plants, along with tetrahydrocannabinol (THC), and accounts for up to 40% of the plant's extract. , clinical research on CBD in ...

Significance

Demand is high for pharmaceutical grade THC and other cannabinoids due to interest in their potential therapeutic use, but is stymied by legal regulations of ''C. sativa'' cultivation in many countries. Direct

chemical synthesis As a topic of chemistry, chemical synthesis (or combination) is the artificial execution of chemical reactions to obtain one or several products. This occurs by physical and chemical manipulations usually involving one or more reactions. In mod ...

of THC is difficult due to high costs and low yields. Therefore, the use of THCA synthase for the production of THC has been explored, as CBGA is easy to synthesize and THCA readily decarboxylates to form THC. Biosynthesis of THCA by expressing THCA synthase in organisms has been attempted in bacteria, insects, and tobacco plants with limited success. Production of THCA on a milligram scale has been demonstrated in ''

Pichia pastoris ''Pichia pastoris'' is a species of methylotrophic yeast. It was found in the 1960s, with its feature of using methanol as a source of carbon and energy. After years of study, ''P. pastoris'' was widely used in biochemical research and biotech ...

'' yeast cells in two independent studies.

References

{{Portal bar, Biology, border=no EC 1.21.3 Tetrahydrocannabinol