Conversion of Substrate Analogs Suggests a Michael Cyclization in Iridoid Biosynthesis

Lindner, Stephanie; Geu‐Flores, Fernando; Bräse, Stefan; Sherden, Nathaniel H.; O’Connor, Sarah E.

doi:10.1016/j.chembiol.2014.09.010

Cited by 35 publications

(46 citation statements)

References 32 publications

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“…4, A-C). The observed products do not readily interconvert under the assay conditions used (40), and the existence of a product mixture is therefore suggestive of a Michael reaction mechanism, as proposed previously for C. roseus CrISY (40). The (24,25).…”

Section: Identification Of Candidate Genes For the Olive Secoiridoidmentioning

confidence: 57%

“…Although iridoids are very common in flowering plants, only in very few cases has iridoid synthase activity been demonstrated in vitro (16,49). Similar to CrISY (16,40), OeISY gave a mixture of nepetalactol and iridodials as enzymatic products. It remains unclear how this mixture of products is channeled into secoiridoid biosynthesis.…”

Section: Discussionmentioning

confidence: 89%

“…Hence, ISY defines a new class of monoterpene cyclase and is mechanistically distinct from canonical terpene cyclases that operate via cationic intermediates. CrISY apparently couples an initial NAD(P)H-dependent 1,4-reduction step with a subsequent Michael-type cyclization (16,40,41).…”

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confidence: 99%

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Identification and Characterization of the Iridoid Synthase Involved in Oleuropein Biosynthesis in Olive (Olea europaea) Fruits

Alagna

Geu‐Flores

Kries

et al. 2016

Journal of Biological Chemistry

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The secoiridoids are the main class of specialized metabolites present in olive (Olea europaea L.) fruit. In particular, the secoiridoid oleuropein strongly influences olive oil quality because of its bitterness, which is a desirable trait. In addition, oleuropein possesses a wide range of pharmacological properties, including antioxidant, anti-inflammatory, and anti-cancer activities. In accordance, obtaining high oleuropein varieties is a main goal of molecular breeding programs. Here we use a transcriptomic approach to identify candidate genes belonging to the secoiridoid pathway in olive. From these candidates, we have functionally characterized the olive homologue of iridoid synthase (OeISY), an unusual terpene cyclase that couples an NAD (P)H-dependent 1,4-reduction step with a subsequent cyclization, and we provide evidence that OeISY likely generates the monoterpene scaffold of oleuropein in olive fruits. OeISY, the first pathway gene characterized for this type of secoiridoid, is a potential target for breeding programs in a high value secoiridoid-accumulating species.Olive (Olea europaea L.) produces a range of secondary metabolites that strongly affect the taste and nutritional properties of olive oil and fruits. The most abundant of these secondary metabolites are the secoiridoids, monoterpenoids with a 3,4-dihydropyran skeleton. These compounds are present as oleosidic secoiridoids or oleosides that have an exocyclic olefinic functionality (1) and possess a tyrosine-derived component (see Fig.

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Section: Identification Of Candidate Genes For the Olive Secoiridoidmentioning

confidence: 57%

Section: Discussionmentioning

confidence: 89%

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Identification and Characterization of the Iridoid Synthase Involved in Oleuropein Biosynthesis in Olive (Olea europaea) Fruits

Alagna

Geu‐Flores

Kries

et al. 2016

Journal of Biological Chemistry

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“…2). Their key role in the cyclisation appears to be to protect the intermediate from general acid catalysed tautomerisation; there is no evidence to suggest that NEPS1/2 mechanisms do not mirror the stepwise Michael addition seen in solution 35 . NEPS3, on the other hand, has specific 7S-cis-cisnepetalactol cyclase activity, binding to 3 possibly via S154 and exerting steric and/or electrostatic influence to enable formation of the cis-cis stereochemistry.…”

Section: Discussionmentioning

confidence: 99%

Uncoupled activation and cyclisation in catmint reductive terpenoid biosynthesis

Lichman

Kamileen

Tichman

et al. 2018

Preprint

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Terpene synthases typically form complex molecular scaffolds by concerted activation and cyclization of linear starting materials in a single enzyme active site. Here we show that iridoid synthase, an atypical reductive terpene synthase, catalyses the activation of its substrate 8-oxogeranial into a reactive enol intermediate but does not catalyse the subsequent cyclisation into nepetalactol. This discovery led us to identify a class of nepetalactol-related short-chain dehydrogenase enzymes (NEPS) from catmint (Nepeta mussinii) which catalyse the stereoselective cyclisation of the enol intermediate into nepetalactol isomers. Subsequent oxidation of nepetalactols by NEPS1 provides nepetalactones, metabolites that are well known for both insect-repellent activity and euphoric effect in cats. Structural characterisation of the NEPS3 cyclase reveals it binds to NAD + yet does not utilise it chemically for a nonoxidoreductive formal [4+2] cyclisation. These discoveries will complement metabolic reconstructions of iridoid and monoterpene indole alkaloid biosynthesis. Main paperNepetalactones 1 are volatile natural products produced by plants of the genus Nepeta, notably catmint (Nepeta mussinii syn racemosa) and catnip (N. cataria) (Fig. 1a) 1,2 . These compounds are responsible for the stimulatory effects these plants have on cats [3][4][5] . Moreover, certain insects use nepetalactones as sex pheromones, so production of these compounds by the plant also impacts interactions with insects 6 . Notably, the bridgehead stereocentres (carbons 4a and 7a) vary between 2 and within 2,4,7 Nepeta species. N. mussinii individuals, for example, produce different ratios of cis-trans 1a, cis-cis 1b and trans-cis-nepetalactone 1c 7 . Variation in stereoisomer ratio may influence the repellence of insect herbivores 8,9 . While the ratio of stereoisomers may be responsible for important biological effects, the mechanism of stereocontrol in nepetalactone biosynthesis is not known.Nepetalactones are iridoids, non-canonical monoterpenoids containing a cyclopentanopyran ring. Canonical cyclic terpenoids (e.g. (-)-limonene) are biosynthesised from linear precursors by terpene synthases (Fig. 1b) 10 . These enzymes activate linear precursors either by loss of pyrophosphate or protonation [10][11][12] . The resulting carbocations generated cyclise rapidly to form an array of cyclic products 13 . Therefore, in canonical terpenoid biosynthesis, activation and cyclisation of precursors are coupled and occur in the same enzyme active site.In plant iridoid biosynthesis, geranyl pyrophosphate is hydrolysed and oxidised into 8-oxogeranial 2 14 . This precursor then undergoes a two-step activation-cyclisation process, analogous to canonical terpene synthesis (Fig. 1c) 15 . Unlike canonical terpene synthesis, however, activation is achieved by reduction, and the intermediate is not a carbocation, but the enol or enolate species 3. Cyclisation of this intermediate yields cis-trans-nepetalactol 4a along with iridodial side products 5 (Fig. 1c).

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“…However, in contrast to secologanin biosynthesis, these pathways are short and do not contain CYP-catalyzed steps. Moreover, secologanin, an iridoid type monoterpene, uses a unique mode of cyclization not observed in canonical terpene biosynthesis (42). Notably, sesquiterpenes, which S. cerevisiae does produce naturally, are produced at much higher levels in a yeast host.…”

Section: Discussionmentioning

confidence: 99%

De novo production of the plant-derived alkaloid strictosidine in yeast

Brown

Clastre

Courdavault

et al. 2015

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

377

328

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The monoterpene indole alkaloids are a large group of plant-derived specialized metabolites, many of which have valuable pharmaceutical or biological activity. There are ∼3,000 monoterpene indole alkaloids produced by thousands of plant species in numerous families. The diverse chemical structures found in this metabolite class originate from strictosidine, which is the last common biosynthetic intermediate for all monoterpene indole alkaloid enzymatic pathways. Reconstitution of biosynthetic pathways in a heterologous host is a promising strategy for rapid and inexpensive production of complex molecules that are found in plants. Here, we demonstrate how strictosidine can be produced de novo in a Saccharomyces cerevisiae host from 14 known monoterpene indole alkaloid pathway genes, along with an additional seven genes and three gene deletions that enhance secondary metabolism. This system provides an important resource for developing the production of more complex plantderived alkaloids, engineering of nonnatural derivatives, identification of bottlenecks in monoterpene indole alkaloid biosynthesis, and discovery of new pathway genes in a convenient yeast host.

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Conversion of Substrate Analogs Suggests a Michael Cyclization in Iridoid Biosynthesis

Cited by 35 publications

References 32 publications

Identification and Characterization of the Iridoid Synthase Involved in Oleuropein Biosynthesis in Olive (Olea europaea) Fruits

Identification and Characterization of the Iridoid Synthase Involved in Oleuropein Biosynthesis in Olive (Olea europaea) Fruits

Uncoupled activation and cyclisation in catmint reductive terpenoid biosynthesis

De novo production of the plant-derived alkaloid strictosidine in yeast

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