Comparative functional analysis of CYP71AV1 natural variants reveals an important residue for the successive oxidation of amorpha‐4,11‐diene

Komori, Aya; Suzuki, Munenori; Seki, Hikaru; Nishizawa, Tomoko; Meyer, J.J.M.; Shimizu, Hideaki; Yokoyama, Shigeyuki; Muranaka, Toshiya

doi:10.1016/j.febslet.2012.11.031

Cited by 23 publications

(25 citation statements)

References 33 publications

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“…1 A, i and Dataset S1), consistent with the reported in vitro activity of CYP71AV1 (13,26,27). Metabolite profiling further revealed a complete loss of all metabolites downstream of A-4,11-D including artemisinin, which typically accumulate in juvenile, expanding and mature WT leaves ( Fig.…”

Section: Resultssupporting

confidence: 84%

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Artemisia annua mutant impaired in artemisinin synthesis demonstrates importance of nonenzymatic conversion in terpenoid metabolism

Czechowski

Larson

Catania

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

101

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Artemisinin, a sesquiterpene lactone produced by Artemisia annua glandular secretory trichomes, is the active ingredient in the most effective treatment for malaria currently available. We identified a mutation that disrupts the amorpha-4,11-diene C-12 oxidase (CYP71AV1) enzyme, responsible for a series of oxidation reactions in the artemisinin biosynthetic pathway. Detailed metabolic studies of cyp71av1-1 revealed that the consequence of blocking the artemisinin biosynthetic pathway is the redirection of sesquiterpene metabolism to a sesquiterpene epoxide, which we designate arteannuin X. This sesquiterpene approaches half the concentration observed for artemisinin in wild-type plants, demonstrating high-flux plasticity in A. annua glandular trichomes and their potential as factories for the production of novel alternate sesquiterpenes at commercially viable levels. Detailed metabolite profiling of leaf maturation time-series and precursor-feeding experiments revealed that nonenzymatic conversion steps are central to both artemisinin and arteannuin X biosynthesis. In particular, feeding studies using 13 C-labeled dihydroartemisinic acid (DHAA) provided strong evidence that the final steps in the synthesis of artemisinin are nonenzymatic in vivo. Our findings also suggest that the specialized subapical cavity of glandular secretory trichomes functions as a location for both the chemical conversion and the storage of phytotoxic compounds, including artemisinin. We conclude that metabolic engineering to produce high yields of novel secondary compounds such as sesquiterpenes is feasible in complex glandular trichomes. Such systems offer advantages over single-cell microbial hosts for production of toxic natural products.artemisinin | p450 oxidase | terpenoid | sesquiterpene | Artemisia annua

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Section: Resultssupporting

confidence: 84%

“…Recent attempts to knock down the A-4,11-D synthase by using RNAi in self-pollinating A. annua resulted in only a modest (30-50%) reduction in artemisinin levels (25). We have chosen to target amorpha-4,11-diene C-12 oxidase (CYP71AV1), which catalyzes the three-step conversion of A-4,11-D to AA (13,26,27).…”

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confidence: 99%

Artemisia annua mutant impaired in artemisinin synthesis demonstrates importance of nonenzymatic conversion in terpenoid metabolism

Czechowski

Larson

Catania

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

101

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“…Finally, comparison of Sh-ASAT3 variants that can add a longer chain to the R 3′ position to those with a preference for short-chain acyl-CoAs revealed that substitution of Cys in place of Tyr at position 41 is sufficient to transform substrate specificity. The comparative genomic/ biochemical approach coupled with in vitro analysis has also been applied to identify key residues of enzymes involved in terpene (48) and artemisinin biosynthesis (49). This approach has the potential to be generally applicable to structure-function studies of any enzymes that make products that are variable across related populations or related species of plants.…”

Section: Discussionmentioning

confidence: 99%

In vitro reconstruction and analysis of evolutionary variation of the tomato acylsucrose metabolic network

Fan

Miller

Schilmiller

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

106

233

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Plant glandular secreting trichomes are epidermal protuberances that produce structurally diverse specialized metabolites, including medically important compounds. Trichomes of many plants in the nightshade family (Solanaceae) produce O-acylsugars, and in cultivated and wild tomatoes these are mixtures of aliphatic esters of sucrose and glucose of varying structures and quantities documented to contribute to insect defense. We characterized the first two enzymes of acylsucrose biosynthesis in the cultivated tomato Solanum lycopersicum. These are type I/IV trichome-expressed BAHD acyltransferases encoded by Solyc12g006330─or S. lycopersicum acylsucrose acyltransferase 1 (Sl-ASAT1)─and Solyc04g012020 (Sl-ASAT2). These enzymes were used-in concert with two previously identified BAHD acyltransferases-to reconstruct the entire cultivated tomato acylsucrose biosynthetic pathway in vitro using sucrose and acyl-CoA substrates.Comparative genomics and biochemical analysis of ASAT enzymes were combined with in vitro mutagenesis to identify amino acids that influence CoA ester substrate specificity and contribute to differences in types of acylsucroses that accumulate in cultivated and wild tomato species. This work demonstrates the feasibility of the metabolic engineering of these insecticidal metabolites in plants and microbes.Solanum | glandular trichomes | acylsugar | specialized metabolism | genotype to phenotype P lants are masters of metabolism, producing hundreds of thousands of small molecules known as specialized metabolites, which vary widely in structure, abundance, and physical and biological properties. These metabolites tend to be produced by enzymes that evolve faster than those that produce "central" metabolites such as amino acids, nucleotides, sugars, and cofactors (1-3), and the pathways and metabolic intermediates involved in biosynthesis of many specialized metabolites remain mysterious. Despite the growing availability of genomic DNA sequences, understanding the genetic and biochemical mechanisms that contribute to this phenotypic diversity and plasticity presents enduring and major challenges in plant biochemistry. It is of great interest to understand and manipulate the biosynthesis of these biologically active molecules.Specialized metabolites typically are produced in a cell-or tissue-specific manner and are generally limited in their taxonomic distribution. Glandular secreting trichomes provide an example of such a differentiated structure; these epidermal "hairs" produce a variety of metabolites of importance to humans, including aromatic flavor components (e.g., in hops for beer and Mediterranean herbs for cooking), psychoactive cannabinoids in Cannabis, and the antimalarial drug artemisinin in Artemisia annua (4, 5).Some trichome-produced metabolites have documented direct and indirect antiherbivore activities (4, 6-8). For example, acylsugars are a group of structurally related specialized metabolites produced in plants of the nightshade family-the Solanaceae (9, 10). Characterized examples ...

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“…Nevertheless, some of the artemisinin biosynthetic enzyme orthologs are expressed in other Artemisia species, including A. absinthium and A. afra, and show similar catalytic activity to those of the corresponding enzymes in A. annua (Komori et al 2013). In this study, we examined the expression of DBR2, the enzyme involved in the branching point step of the artemisinin biosynthetic pathway, in A. absinthium and A. afra.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, we investigated the expression of genes involved in artemisinin biosynthesis in these Artemisia species and analyzed the function of orthologous enzymes in a yeast expression system. We found that CYP71AV1 orthologs were expressed in A. absinthium (abCYP71AV1) and A. afra (afCYP71AV1) and showed similar catalytic activity to A. annua CYP71AV1 (Komori et al 2013).…”

Section: Introductionmentioning

confidence: 92%

Functional analysis of orthologous artemisinic aldehyde Δ11(13)-reductase reveals potential artemisinin-producing activity in non-artemisinin-producing <i>Artemisia absinthium</i>

Muangphrom

Suzuki

Seki

et al. 2014

Plant Biotechnology

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Artemisinin is the most effective antimalarial compound isolated from Artemisia annua. Artemisinic aldehyde Δ11(13)-reductase (DBR2) catalyzes the reduction of artemisinic aldehyde into dihydroartemisinic aldehyde, switching the pathway towards artemisinin production. Although other Artemisia species cannot produce artemisinin, we found a putative DBR2 ortholog expressed in A. absinthium (abDBR2). We examined the catalytic activity of abDBR2 in vitro and found that it shows comparable activity to that of DBR2 based on the reduction of artemisinic aldehyde into dihydroartemisinic aldehyde. Furthermore, we found that dihydroartemisinic aldehyde was detected in the extract of A. absinthium leaves fed with artemisinic aldehyde, suggesting the presence of active abDBR2 in planta. Our results indicate that A. absinthium may be a potential host for the production of artemisinin through metabolic engineering.

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Comparative functional analysis of CYP71AV1 natural variants reveals an important residue for the successive oxidation of amorpha‐4,11‐diene

Cited by 23 publications

References 33 publications

Artemisia annua mutant impaired in artemisinin synthesis demonstrates importance of nonenzymatic conversion in terpenoid metabolism

Artemisia annua mutant impaired in artemisinin synthesis demonstrates importance of nonenzymatic conversion in terpenoid metabolism

In vitro reconstruction and analysis of evolutionary variation of the tomato acylsucrose metabolic network

Functional analysis of orthologous artemisinic aldehyde Δ11(13)-reductase reveals potential artemisinin-producing activity in non-artemisinin-producing <i>Artemisia absinthium</i>

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Comparative functional analysis of CYP71AV1 natural variants reveals an important residue for the successive oxidation of amorpha‐4,11‐diene

Cited by 23 publications

References 33 publications

Artemisia annua mutant impaired in artemisinin synthesis demonstrates importance of nonenzymatic conversion in terpenoid metabolism

Artemisia annua mutant impaired in artemisinin synthesis demonstrates importance of nonenzymatic conversion in terpenoid metabolism

In vitro reconstruction and analysis of evolutionary variation of the tomato acylsucrose metabolic network

Functional analysis of orthologous artemisinic aldehyde &#916;11(13)-reductase reveals potential artemisinin-producing activity in non-artemisinin-producing <i>Artemisia absinthium</i>

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Functional analysis of orthologous artemisinic aldehyde Δ11(13)-reductase reveals potential artemisinin-producing activity in non-artemisinin-producing <i>Artemisia absinthium</i>