Structural determinants of reductive terpene cyclization in iridoid biosynthesis

Kries, Hajo; Caputi, Lorenzo; Stevenson, Clare E. M.; Kamileen, Mohamed O.; Sherden, Nathaniel H.; Geu‐Flores, Fernando; Lawson, David M.; O’Connor, Sarah E.

doi:10.1038/nchembio.1955

Cited by 59 publications

(93 citation statements)

References 34 publications

(38 reference statements)

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“…It is possible that the flexibility of the NR active site loop can aid in the proper orientation of the substrate for catalysis. A similar role for the active site loop as an adaptive feature for allowing the binding of different substrates has been proposed for the SDRs that catalyze reductive terpene cyclization in iridoid alkaloid biosynthesis (36).…”

Section: Discussionmentioning

confidence: 99%

Identification of a Noroxomaritidine Reductase with Amaryllidaceae Alkaloid Biosynthesis Related Activities

Kilgore

Holland

Jez

et al. 2016

Journal of Biological Chemistry

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Amaryllidaceae alkaloids are a large group of plant natural products with over 300 documented structures and diverse biological activities. Several groups of Amaryllidaceae alkaloids including the hemanthamine-and crinine-type alkaloids show promise as anticancer agents. Two reduction reactions are required for the production of these compounds: the reduction of norcraugsodine to norbelladine and the reduction of noroxomaritidine to normaritidine, with the enantiomer of noroxomaritidine dictating whether the derivatives will be the crinine-type or hemanthamine-type. It is also possible for the carbon-carbon double bond of noroxomaritidine to be reduced, forming the precursor for maritinamine or elwesine depending on the enantiomer reduced to an oxomaritinamine product. In this study, a short chain alcohol dehydrogenase/reductase that co-expresses with the previously discovered norbelladine 4-Omethyltransferase from Narcissus sp. and Galanthus spp. was cloned and expressed in Escherichia coli. Biochemical analyses and x-ray crystallography indicates that this protein functions as a noroxomaritidine reductase that forms oxomaritinamine from noroxomaritidine through a carbon-carbon double bond reduction. The enzyme also reduces norcraugsodine to norbelladine with a 400-fold lower specific activity. These studies identify a missing step in the biosynthesis of this pharmacologically important class of plant natural products.

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Section: Discussionmentioning

confidence: 99%

Identification of a Noroxomaritidine Reductase with Amaryllidaceae Alkaloid Biosynthesis Related Activities

Kilgore

Holland

Jez

et al. 2016

Journal of Biological Chemistry

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“…Structural and mechanistic studies of ISY from C. roseus have revealed that the key active site residue is Tyr178 (37,39). This residue is also conserved in all ISY homologs identified from Nepeta (Figure 3).…”

Section: Sequence Of Nepeta Isymentioning

confidence: 99%

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

Sherden

Lichman

Caputi

et al. 2017

Preprint

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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)

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“…The enzymatic control of the initial reductive activation step has been structurally characterised in CrISY: crystal structures with cofactor and inhibitor or substrate show binding modes conducive to reduction and formation of an enolate intermediate [21][22][23] . Furthermore, this reduction is stereoselective, as exemplified by the comparison of CrISY, which produces 7S-4a, with AmISY, which produces the enantiomer 18 .…”

Section: Figurementioning

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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Structural determinants of reductive terpene cyclization in iridoid biosynthesis

Cited by 59 publications

References 34 publications

Identification of a Noroxomaritidine Reductase with Amaryllidaceae Alkaloid Biosynthesis Related Activities

Identification of a Noroxomaritidine Reductase with Amaryllidaceae Alkaloid Biosynthesis Related Activities

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

Uncoupled activation and cyclisation in catmint reductive terpenoid biosynthesis

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