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

Alagna, Fiammetta; Geu‐Flores, Fernando; Kries, Hajo; Panara, Francesco; Baldoni, Luciana; O’Connor, Sarah E.; Osbourn, Anne

doi:10.1074/jbc.m115.701276

Cited by 70 publications

(69 citation statements)

References 57 publications

(72 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…ISY was first discovered in Catharanthus roseus (CrISY) where it forms part of the biosynthetic route to the anti-cancer monoterpene indole alkaloids vincristine and vinblastine 15,16 . Subsequent studies revealed ISYs from Olive (Olea europaea, OeISY) 17 and Snapdragon (Antirrhinum majus, AmISY) 18 , as well as paralogous proteins in C. roseus with promiscuous ISY activity 19 . Recently, we identified ISYs from Nepeta 20 .…”

Section: Figurementioning

confidence: 99%

Uncoupled activation and cyclisation in catmint reductive terpenoid biosynthesis

Lichman

Kamileen

Tichman

et al. 2018

Preprint

View full text Add to dashboard Cite

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).

show abstract

Section: Figurementioning

confidence: 99%

Uncoupled activation and cyclisation in catmint reductive terpenoid biosynthesis

Lichman

Kamileen

Tichman

et al. 2018

Preprint

View full text Add to dashboard Cite

show abstract

“…http://dx.doi.org/10.1101/179572 doi: bioRxiv preprint first posted online Aug. 22, 2017; 8 of ISY, from C. roseus (30) and Olea europaea (olive) (36), that have been functionally characterized were used to search the Nepeta transcriptomes for ISY homologs using BLAST. Both Nepeta species each contained two distinct homologs of ISY, which were named family 1 and family 2 (amino acid sequence identity between N. cataria family 1 (NcISY1) and N. cataria family 2 (NcISY2) was 80%; between N. mussinii family 1 (NmISY1) and family 2 (NmISY2) was 79%).…”

Section: Cloning Heterologous Expression and Biochemical Assay Of Ismentioning

confidence: 99%

Identification of Iridoid Synthases from Nepeta species: Iridoid cyclization does not determine nepetalactone stereochemistry

Sherden

Lichman

Caputi

et al. 2017

Preprint

View full text Add to dashboard Cite

Iridoid cyclization does not determine nepetalactone stereochemistry Graphical AbstractIridoid synthase from Nepeta cateria (catnip) and Nepeta mussinii, have been cloned and characterized. O OH H H O O 7α 4αpeer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/179572 doi: bioRxiv preprint first posted online peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.Thecopyright holder for this preprint (which was not . http://dx.doi.org/10.1101/179572 doi: bioRxiv preprint first posted online Aug. 22, 2017; 3 AbstractNepetalactones are iridoid monoterpenes with a broad range of biological activities produced by plants in the Nepeta genus. However, none of the genes for nepetalactone biosynthesis have been discovered. Here we report the transcriptomes of two Nepeta species, each with distinctive profiles of nepetalactone stereoisomers. As a starting point for investigation of nepetalactone biosynthesis in Nepeta, these transcriptomes were used to identify candidate genes for iridoid synthase homologs, an enzyme that has been shown to form the core iridoid skeleton in several iridoid producing plant species. Iridoid synthase homologs identified from the transcriptomes were cloned, heterologously expressed, and then assayed with the 8-oxogeranial substrate. These experiments revealed that catalytically active iridoid synthase enzymes are present in Nepeta, though there are unusual mutations in key active site residues. Nevertheless, these enzymes exhibit similar catalytic activity and product profile compared to previously reported iridoid synthases from other plants. Notably, four nepetalactone stereoisomers with differing stereochemistry at the 4a and 7a positions -which are generated during the iridoid synthase reaction -are observed at different ratios in various Nepeta species. This work strongly suggests that the variable stereochemistry at these 4a and 7a positions of nepetalactone diastereomers is established further downstream in the iridoid pathway in Nepeta. Overall, this work provides a gateway into the biosynthesis of nepetalactones in Nepeta. peer-reviewed)

show abstract

“…Oleuropein is a metabolite of high interest for humans as it exhibits antioxidant, antiinflammatory, antiproliferative, antimicrobic and antiviral activity (Bulotta et al, 2013). Apart from a recently identified iridoid synthase (Alagna et al, 2016), other enzymes involved in the biosynthesis of oleuropein are unknown. This metabolite is stored in a glycosylated and thus inactive form, but upon cell disruption (i.e.…”

mentioning

confidence: 99%

The C-Domain of Oleuropein β-Glucosidase Assists in Protein Folding and Sequesters the Enzyme in Nucleus

et al. 2017

View full text Add to dashboard Cite

Oleuropein, a terpene-derived glycosylated secoiridoid biosynthesized exclusively by members of the Oleaceae family, is involved in a two-component defense system comprising a b-glucosidase that activates oleuropein into a toxic glutaraldehydelike structure. Oleuropein and its deglycosylated derivatives have high pharmaceutical interest. In this study we determined that the in planta heterologous expressed OeGLU, an oleuropein-specific b-glucosidase from olive (Olea europaea), had enzymatic kinetics similar to the olive native enzyme. The C terminus encompassing the nuclear localization signal sequesters the enzyme in the nucleus, and predetermines the protein-protein recognition and homodimerization. Biochemical analysis revealed that OeGLU is a homomultimer with high M r . In silico prediction modeling of the complex structure and bimolecular fluorescence complementation analyses revealed that the C terminus of OeGLU is essential for the proper assembly of an octameric form, a key conformational feature that determines the activity of the enzyme. Our results demonstrate that intrinsic characteristics of the OeGLU ensure separation from oleuropein and keep the dual-partner defensive system conditionally inactive. Upon cell destruction, the dual-partner defense system is activated and olive massively releases the arsenal of defense.

show abstract

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

Cited by 70 publications

References 57 publications

Uncoupled activation and cyclisation in catmint reductive terpenoid biosynthesis

Uncoupled activation and cyclisation in catmint reductive terpenoid biosynthesis

Identification of Iridoid Synthases from Nepeta species: Iridoid cyclization does not determine nepetalactone stereochemistry

The C-Domain of Oleuropein β-Glucosidase Assists in Protein Folding and Sequesters the Enzyme in Nucleus

Contact Info

Product

Resources

About