The

enzyme An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...

amorpha-4,11-diene synthase (EC 4.2.3.24) (ADS) catalyzes the

chemical reaction A chemical reaction is a process that leads to the chemistry, chemical transformation of one set of chemical substances to another. When chemical reactions occur, the atoms are rearranged and the reaction is accompanied by an Gibbs free energy, ...

:(2''E'',6''E'')-farnesyl diphosphate ⇌ amorpha-4,11-diene + diphosphate This enzyme belongs to the family of

lyase In biochemistry, a lyase is an enzyme that catalyzes the breaking (an elimination reaction) of various chemical bonds by means other than hydrolysis (a substitution reaction) and oxidation Redox ( , , reduction–oxidation or oxidatio ...

s, specifically those carbon-oxygen lyases acting on phosphates. The

systematic name A systematic name is a name given in a systematic way to one unique group, organism, object or chemical substance, out of a specific population or collection. Systematic names are usually part of a nomenclature. A semisystematic name or semitrivi ...

of this enzyme class is (2''E'',6''E'')-farnesyl-diphosphate diphosphate-lyase (amorpha-4,11-diene-forming). This enzyme is also called amorphadiene synthase. This enzyme is mainly found in '' Artemisia annua'', a temperate Asian native flowering plant, and ADS catalyzes the first committed step in the antimalarial drug artemisinin synthesis.

Enzyme Properties

Physical Properties

Amorpha-4,11-diene synthase is a 533 amino acid long protein with a molecular weight of 62.2 kDa and

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

of 5.25. ADS shows a pH optimum at pH 6.5 and a minimum at pH 7.5. With Mg²⁺, Mn²⁺ and Co²⁺ as cofactors, large enzyme activity observed, with Ni²⁺, low activity observed, and with Cu²⁺ and Zn²⁺, essentially no activity observed.

Evolution

ADS is a highly conserved protein similar to other proteins with analogous functionality. The deduced amino acid sequence is 32 to 51% identical with the sequence of other known sesquiterpene cyclases from

angiosperms Flowering plants are plants that bear flowers and fruits, and form the clade Angiospermae (). The term angiosperm is derived from the Greek words (; 'container, vessel') and (; 'seed'), meaning that the seeds are enclosed within a fruit. T ...

(flowering plants) meaning the enzymes have a common ancestry. More specifically, it has a highly conserved substrate binding site with an

aspartate Aspartic acid (symbol Asp or D; the ionic form is known as aspartate), is an α-amino acid that is used in the biosynthesis of proteins. The L-isomer of aspartic acid is one of the 22 proteinogenic amino acids, i.e., the building blocks of protein ...

rich DDxxD motif.

Expression

ADS is expressed 16-fold higher in the leaves than in roots of the '' Artemisia annua'' plant and 10-fold higher than in the stems showing a tissue-specific expression pattern.

Products

While amorpha-4,11-diene is the main project of ADS, the purified enzyme has been shown to produce at least 16 different products. These additional products include the olefins (E)-β-farnesene, amorpha-4,7(11)-diene, γ-humulene and β-sesquiphellandrene, and the oxygenated sesquiterpenes amorpha-4-en-11-ol, amorpha-4-en-7-ol, and α-bisabolol. About 97.5% of the products are

olefins In organic chemistry, an alkene, or olefin, is a hydrocarbon containing a carbon–carbon double bond. The double bond may be internal or at the terminal position. Terminal alkenes are also known as α-olefins. The International Union of P ...

and the other 2.5% are oxygenated sesquiterpenes.

Regulation

Two forms of ADS regulation include environmental induction and biochemical regulatory switches. Under normal conditions, ADS is expressed at low levels in '' Artemisia annua''; however, when exposed to cold, heat shock, or UV light, the ADS becomes upregulated. Corresponding with this in nature, cold-acclimated ''Artemisia annua'' express higher levels of ADS than plants under normal conditions. Regulatory switches help control levels of ADS. Since enzyme substrate Farnesyl diphosphate has many uses in addition to forming amorpha-4,11-diene, these other pathways regulate ADS. One such pathway is sterol biosynthesis, and in fact, the enzyme

squalene synthase Squalene synthase (SQS) or farnesyl-diphosphate:farnesyl-diphosphate farnesyl transferase is an enzyme localized to the membrane of the endoplasmic reticulum. SQS participates in the terpenoid, isoprenoid biosynthetic pathway, catalyzing a two-s ...

(SS) is considered a regulatory switch for ADS. When SS

cDNA In genetics, complementary DNA (cDNA) is DNA that was reverse transcribed (via reverse transcriptase) from an RNA (e.g., messenger RNA or microRNA). cDNA exists in both single-stranded and double-stranded forms and in both natural and engin ...

, which reduces SS

mRNA In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of Protein biosynthesis, synthesizing a protein. mRNA is ...

concentration and therefore reduces expression of SS, is introduced into the plant cells, mRNA levels of ADS dramatically increased (Figure 1).

Mechanism

ADS catalyzes the reaction of farnesyl diphosphate(FPP) to amorpha-4,11-diene (Figure 2). The following reaction mechanism has been supported with experimental data: #

Isomerization In chemistry, isomerization or isomerisation is the process in which a molecule, polyatomic ion or molecular fragment is transformed into an isomer with a different chemical structure. Enolization is an example of isomerization, as is tautomer ...

of FPP to (R)-nerolidyl diphosphate (NPP) # Ionization of NPP # C-1,C-6-ring closure to generate a bisaboyl cation # 1,3-hydride shift # 1,10-ring closure # Deprotonation at either C-12 or C-13

Industrial applications

Amorpha-4-11-diene synthase catalyses the first step in the synthesis of antimalarial drug artemisinin by converting ubiquitous farnesyl diphosphate into the precursor amorpha-4,11-diene. Armorpha-4,11-diene undergoes multiple steps to become artemisinic acid and finally artemisinin (Figure 3). Artemisinin is naturally synthesized by the native Chinese plant '' Artemisia annua''; however due to low plant tissue extraction yields and long growing seasons, alternative ways including metabolic engineering to produce artemisinin are being considered. ADS has been cloned and expressed in bacteria cells as a way to produce artemisinin. Because ADS is the first committed and limiting step of artemisinin biosynthesis, increasing ADS levels should increase artemisinin yield. However, ADS is not the only bottle neck in artemisinin production, so additional genes are needed to increase yield. A way to do this has been to increase the flux towards ADS by producing more FPP from the mevalonate pathway. In fact, the over-expression of Amorpha-4-11-diene synthase coupled with expression of yeast's mevalonate pathway has shown to increase yield and production of artemisinin precursor amorpha-4,11-diene. Scientists from the company Amyris have developed a method for high-level production of artemisinin. One of the synthetic genes in this procedure is ADS from ''Artemisia annua''. The semi-synthetic production of artemisinin by Amyris has the potential to lower the cost of antimalarial treatments thus making them more readily available to the developing world.

References

{{Portal bar, Biology, border=no EC 4.2.3 Enzymes of unknown structure