bisabolol and amorpha-4-11-diene

The production of artemisinin, the most effective antimalarial compound, is limited to Artemisia annua. Enzymes involved in artemisinin biosynthesis include amorpha-4,11-diene synthase (ADS), amorpha-4,11-diene 12-monooxygenase (CYP71AV1) and artemisinic aldehyde Δ(11)13 reductase (DBR2). Although artemisinin and its specific intermediates are not detected in other Artemisia species, we reported previously that CYP71AV1 and DBR2 homologs were expressed in some non-artemisinin-producing Artemisia plants. These homologous enzymes showed similar functions to their counterparts in A. annua and can convert fed intermediates into the following products along the artemisinin biosynthesis in planta These findings suggested a partial artemisinin-producing ability in those species. In this study, we examined genes highly homologous to ADS, the first committed gene in the pathway, in 13 Artemisia species. We detected ADS homologs in A. absinthium, A. kurramensis and A. maritima. We analyzed the enzymatic functions of all of the ADS homologs after obtaining their cDNA. We found that the ADS homolog from A. absinthium exhibited novel activity in the cyclization of farnesyl pyrophosphate (FPP) to koidzumiol, a rare natural sesquiterpenoid. Those from A. kurramensis and A. maritima showed similar, but novel, activities in the cyclization of FPP to (+)-α-bisabolol. The unique functions of the novel sesquiterpene synthases highly homologous to ADS found in this study could provide insight into the molecular basis of the exceptional artemisinin-producing ability in A. annua.

Topics: Alkyl and Aryl Transferases; Amino Acid Sequence; Antimalarials; Artemisia; Artemisia annua; Artemisinins; Biosynthetic Pathways; Fabaceae; Gene Expression Regulation, Plant; Lactones; Mixed Function Oxygenases; Monocyclic Sesquiterpenes; Oxidoreductases; Phylogeny; Plant Proteins; Polycyclic Sesquiterpenes; Polyisoprenyl Phosphates; Sequence Alignment; Sesquiterpenes

Amorphadiene synthase (ADS) is known for its vital role as a key enzyme in the biosynthesis of the antimalarial drug artemisinin. Despite the vast research targeting this enzyme, an X-ray crystal structure of the enzyme has not yet been reported. In spite of the remarkable difference in product profile among various sesquiterpene synthases, they all share a common α-helical fold with many highly conserved regions especially the bivalent metal ion binding motifs. Hence, to better understand the structural basis of the mechanism of ADS, a reliable 3D homology model representing the conformation of the ADS enzyme and the position of its substrate, farnesyl diphosphate, in the active site was constructed. The model was generated using the reported crystal structure of α-bisabolol synthase mutant, an enzyme with high sequence identity with ADS, as a template. Site-directed mutagenesis was used to probe the active site residues. Seven residues were probed showing their vital role in the ADS mechanism and/or their effect on product profile. The generated variants confirmed the validity of the ADS model. This model will serve as a basis for exploring structure-function relationships of all residues in the active site to obtain further insight into the ADS mechanism.

Topics: Alkyl and Aryl Transferases; Artemisinins; Catalytic Domain; Fabaceae; Gas Chromatography-Mass Spectrometry; Molecular Structure; Monocyclic Sesquiterpenes; Polycyclic Sesquiterpenes; Sesquiterpenes