Iridoid Synthase Research Articles

The genomic and enzymatic basis for iridoid biosynthesis in cat thyme (Teucrium marum).

Iridoids are non-canonical monoterpenoids produced by both insects and plants. An example is the cat-attracting and insect-repelling volatile iridoid nepetalactone, produced by Nepeta sp. (catmint) and aphids. Recently, both nepetalactone biosynthetic pathways were elucidated, showing a remarkable convergent evolution. The iridoid, dolichodial, produced by Teucrium marum (cat thyme) and multiple insect species, has highly similar properties to nepetalactone but its biosynthetic origin remains unknown. We set out to determine the genomic, enzymatic, and evolutionary basis of iridoid biosynthesis in T. marum. First, we generated a de novo chromosome-scale genome assembly for T. marum using Oxford Nanopore Technologies long reads and proximity-by-ligation Hi-C reads. The 610.3 Mb assembly spans 15 pseudomolecules with a 32.9 Mb N50 scaffold size. This enabled identification of iridoid biosynthetic genes, whose roles were verified via activity assays. Phylogenomic analysis revealed that the evolutionary history of T. marum iridoid synthase, the iridoid scaffold-forming enzyme, is not orthologous to typical iridoid synthases but is derived from its conserved paralog. We discovered an enzymatic route from nepetalactol to diverse iridoids through the coupled activity of an iridoid oxidase cytochrome P450 and acetyltransferases, via an inferred acylated intermediate. This work provides a genomic resource for specialized metabolite research in mints and demonstration of the role of acetylation in T. marum iridoid diversity. This work will enable future biocatalytic or biosynthetic production of potent insect repellents, as well as comparative studies into iridoid biosynthesis in insects.

A fused hybrid enzyme of 8-hydroxygeraniol oxidoreductase (8HGO) from Gardenia jasminoides and iridoid synthase (ISY) from Catharanthus roseus significantly enhances nepetalactol and iridoid production.

The operation of 8HGO-ISY fusion enzymes can increase nepetalactol flux to iridoid biosynthesis, and the Gj8HGO-CrISY expression in Gardenia jasminoides indicates that seco-iridoids and closed-ring iridoids share a nepetalactol pool. Nepetalactol is a common precursor of (seco)iridoids and their derivatives, which are a group of noncanonical monoterpenes. Functional characterization of an 8HGO (8-hydroxygeraniol oxidoreductase) from Catharanthus roseus, a seco-iridoids producing plant, has been reported; however, the 8HGO from G. jasminoides with plenty of closed-ring iridoids remains uninvestigated. In this work, a Gj8HGO was cloned and biochemically characterized. In addition, the relatively low production of nepetalactol in plants and engineered microbial host is likely to be attributed to the fact that Cr8HGO and CrISY (iridoid synthase) are substrate-promiscuous enzymes catalyzing unexpected substrates to the undesired products. Herein, a bifunctional enzyme consisting of an 8HGO fused to an ISY was designed for the proximity to the substrate and recycling of NADP+ and NADPH cofactor to reduce the undesired intermediate in the synthesis of nepetalactol. Of four fusion enzymes (i.e., Gj8HGO-GjISY, Gj8HGO-GjISY2, Gj8HGO-GjISY4, and Gj8HGO-CrISY), interestingly, only the last one can enable cascade reaction to form cis-trans-nepetalactol. Furthermore, we establish a reliable Agrobacterium-mediated transformation system. The expression of Gj8HGO-CrISY in G. jasminoides led to a significant enhancement of nepetalactol production, about 19-fold higher than that in wild-type plants, which further resulted in the twofold to fivefold increase of total iridoids and representative iridoid such as geniposide, indicating that seco-iridoids in C. roseus and closed-ring iridoids in G. jasminoides share a nepetalactol pool. All results suggest that 8HGO and ISY can be manipulated to maximize metabolic flux for nepetalactol and iridoid production.

Functional iridoid synthases from iridoid producing and non-producing Nepeta species (subfam. Nepetoidae, fam. Lamiaceae).

Iridoids, a class of atypical monoterpenes, exhibit exceptional diversity within the Nepeta genus (subfam. Nepetoidae, fam. Lamiaceae).The majority of these plants produce iridoids of the unique stereochemistry, with nepetalactones (NLs) predominating; however, a few Nepeta species lack these compounds. By comparatively analyzing metabolomics, transcriptomics, gene co-expression, and phylogenetic data of the iridoid-producing N. rtanjensis Diklić & Milojević and iridoid-lacking N. nervosa Royle & Bentham, we presumed that one of the factors responsible for the absence of these compounds in N. nervosa is iridoid synthase (ISY). Two orthologues of ISY were mined from leaves transcriptome of N. rtanjensis (NrPRISE1 and NrPRISE2), while in N. nervosa only one (NnPRISE) was identified, and it was phylogenetically closer to the representatives of the Family 1 isoforms, designated as P5βRs. Organ-specific and MeJA-elicited profiling of iridoid content and co-expression analysis of IBG candidates, highlighted NrPRISE2 and NnPRISE as promising candidates for ISY orthologues, and their function was confirmed using in vitro assays with recombinant proteins, after heterologous expression of recombinant proteins in E. coli and their His-tag affinity purification. NrPRISE2 demonstrated ISY activity both in vitro and likely in planta, which was supported by the 3D modeling and molecular docking analysis, thus reclassification of NrPRISE2 to NrISY is accordingly recommended. NnPRISE also displays in vitro ISY-like activity, while its role under in vivo conditions was not here unambiguously confirmed. Most probably under in vivo conditions the NnPRISE lacks substrates to act upon, as a result of the loss of function of some of the upstream enzymes of the iridoid pathway. Our ongoing work is conducted towards re-establishing the biosynthesis of iridoids in N. nervosa.

Transcriptome‐based identification and functional characterization of iridoid synthase involved in monotropein biosynthesis in blueberry

AbstractBlueberries (Vaccinium spp.) are well known for their nutritional quality, and recent work has shown that Vaccinium spp. also produce iridoids, which are specialized metabolites with potent health‐promoting benefits. The iridoid glycoside monotropein, which has anti‐inflammatory and antinociceptive activities, has been detected in several wild blueberry species but in only a few cultivated highbush blueberry cultivars. How monotropein is produced in blueberry and the genes involved in its biosynthesis remain to be elucidated. Using a monotropein‐positive (M+) and monotropein‐negative (M−) cultivar of blueberry, we employed transcriptomics and comparative genomics to identify candidate genes in the blueberry iridoid biosynthetic pathway. Orthology analysis was completed using de novo transcript assemblies for both the M+ and M− blueberry cultivars along with the known iridoid‐producing plant species Catharanthus roseus to identify putative genes involved in key steps in the early iridoid biosynthetic pathway. From the identified orthologs, we functionally characterized iridoid synthase (ISY), a key enzyme involved in formation of the iridoid scaffold, from both the M+ and M− cultivars. Detection of nepetalactol suggests that ISY from both the M+ and M− cultivars produce functional enzymes that catalyze the formation of iridoids. Transcript accumulation of the putative ISY gene did not correlate with monotropein production, suggesting other genes in the monotropein biosynthetic pathway may be more directly responsible for differential accumulation of the metabolite in blueberry. Mutual rank analysis revealed that ISY is co‐expressed with UDP‐glucuronosyltransferase, which encodes an enzyme downstream of the ISY step. Results from this study contribute new knowledge in our understanding of iridoid biosynthesis in blueberry and could lead to development of new cultivars with increased human health benefits.

Exploring the Multitarget Potential of Iridoids: Advances and Applications.

Iridoids are secondary plant metabolites that are multitarget compounds active against various diseases. Iridoids are structurally classified into iridoid glycosides and non-glycosidic iridoids according to the presence or absence of intramolecular glycosidic bonds; additionally, iridoid glycosides can be further subdivided into carbocyclic iridoids and secoiridoids. These monoterpenoids belong to the cyclopentan[c]-pyran system, which has a wide range of biological activities, including antiviral, anticancer, antiplasmodial, neuroprotective, anti-thrombolytic, antitrypanosomal, antidiabetic, hepatoprotective, anti-oxidant, antihyperlipidemic and anti-inflammatory properties. The basic chemical structure of iridoids in plants (the iridoid ring scaffold) is biosynthesized in plants by the enzyme iridoid synthase using 8-oxogeranial as a substrate. With advances in phytochemical research, many iridoid compounds with novel structure and outstanding activity have been identified in recent years. Biologically active iridoid derivatives have been found in a variety of plant families, including Plantaginaceae, Rubiaceae, Verbenaceae, and Scrophulariaceae. Iridoids have the potential of modulating many biological events in various diseases. This review highlights the multitarget potential of iridoids and includes a compilation of recent publications on the pharmacology of iridoids. Several in vitro and in vivo models used, along with the results, are also included in the paper. This paper's systematic summary was created by searching for relevant iridoid material on websites such as Google Scholar, PubMed, SciFinder Scholar, Science Direct, and others. The compilation will provide the researchers with a thorough understanding of iridoid and its congeners, which will further help in designing a large number of potential compounds with a strong impact on curing various diseases.

Bacillus H47 triggers Olea europaea metabolism activating DOXP and shikimate pathways simultaneously and modifying leaf extracts' antihypertensive activity.

Improvement of plant adaptation by beneficial bacteria (PGPB) may be achieved by triggering multiple pathways to overcome the environmental stress on plant’s growth cycle, activating plant’s metabolism. The present work reports the differential ability of three Bacillus strains to trigger olive tree metabolism, among which, only H47 was outstanding increasing iridoid and flavonol concentration. One-year old olive seedlings grown open air, under harsh conditions of water shortage in saline soils, were root-inoculated with three Bacillus PGPB strains throughout a 12-month period after which, photosynthesis was determined; photosynthetic pigments and bioactive secondary metabolites (iridoids and flavonols) were analyzed, and a study of gene expression of both pathways involved was undertaken to unravel molecular targets involved in the activation. All three strains increased plant fitness based on photosynthetic values, increasing energy dissipation capacity to lower oxidative stress; only H47 increased CO2 fixation and transpiration. Bacillus H47 was found to trigger synthases in the DOXP pathway (up to 5-fold in DOXP-synthase, 3.5-fold in Iridoid synthase, and 2-fold in secologanin synthase) associated to a concomitant increase in iridoids (up to 5-fold in oleuropein and 2-fold in its precursor secologanin). However, despite the 2-fold increases detected in the two predominant flavonols, gene expression was not enhanced, suggesting involvement of a pulse activation model proposed for innate immunity. Furthermore, the activity of leaf extracts to inhibit Angiotensin Converting Enzyme was evaluated, to explore further uses of plant debris with higher added value. Despite the increases in iridoids, leaf extracts from H47 did not increase ACE inhibition, and still, increased antihypertensive potential in oil obtained with this strain is to be explored, as leaves are the source for these metabolites which further translocate to fruits. In summary, Bacillus H47 is an effective strain to increase plant adaptation to dry and saline environments, activates photosynthesis and secondary metabolism in olive tree.

Identification and functional characterization of three iridoid synthases in Gardenia jasminoides.

The discovery of three iridoid synthases (GjISY, GjISY2 and GjISY4) from Gardenia jasminoides and their functional characterization increase the understanding of iridoid scaffold/iridoid glycoside biosynthesis in iridoid-producing plants. Iridoids are a class of noncanonical monoterpenes that are found naturally in the plant kingdom mostly as glycosides. Over 40 iridoid glycosides (e.g., geniposide, gardenoside and shanzhiside) have been isolated from Gardenia jasminoides. They have multiple pharmacological properties and health-promoting effects. However, their biosynthetic pathway is poorly understood, and the iridoid synthase (ISY) responsible for the cyclization of the core scaffold remains unclear. In this study, three homologs of ISYs from G. jasminoides (GjISY, GjISY2 and GjISY4) were identified on the basis of transcriptomic data and functionally characterized. The genomic structure and intron-exon arrangement revealed that all three ISYs contained an intron. Biochemical assays indicated that all three recombinant enzymes reduced 8-oxogeranial to nepetalactol and its open forms (iridodials) as the products of the classical CrISY (Catharanthus roseus). In addition, all three enzymes reduced progesterone to 5-β-prognane-3,20-dione. However, only GjISY2 and GjISY4 reduced 2-cyclohexen-1-one to cyclohexanone. Overall, the GjISY2 expression levels in the flowers and fruits were similar to the GjISY and GjISY4 expression levels. By contrast, the GjISY2 expression levels in the upper and lower leaves were substantially higher than the GjISY and GjISY4 expression levels. Among the three, GjISY2 exhibited the highest catalytic efficiency for 8-oxogeranial. GjISY2 might be the major contributor to iridoid biosynthesis in G. jasminoides. Collectively, our results advance the understanding of iridoid scaffold/iridoid glycoside biosynthesis in G. jasminoides and provide a potential target for metabolic engineering and breeding.

A “push-pull-restrain” strategy to improve citronellol production in Saccharomyces cerevisiae

Microbial production of monoterpenes has attracted increasing attention in recent years. Up to date, there are only few reports on the biosynthesis of the monoterpene alcohol citronellol that is widely used as fragrant and pharmaceutical intermediates. Here, we engineered Saccharomyces cerevisiae by employing a “push-pull-restrain” strategy to improve citronellol production based on the reduction of geraniol. Starting from a engineered geraniol-producing strain, different reductases were investigated and the best performing iridoid synthase from Catharanthus roseus (CrIS) resulted in 285.89 mg/L enantiomerically pure S-citronellol in shake flasks. Geranyl diphosphate (GPP), the most important precursor for monoterpenes, was enhanced by replacing the wild farnesyl diphosphate synthase (Erg20) with the mutant Erg20F96W, increasing the citronellol titer to 406.01 mg/L without negative influence on cell growth. Moreover, we employed synthetic protein scaffolds and protein fusion to colocalize four sequential enzymes to achieve better substrate channeling along with the deletion of an intermediate degradation pathway gene ATF1, which elevated the citronellol titer to 972.02 mg/L with the proportion of 97.8% of total monoterpenes in YPD medium. Finally, the engineered strain with complemented auxotrophic markers produced 8.30 g/L of citronellol by fed-batch fermentation, which was the highest citronellol titer reported to date. The multi-level engineering strategies developed here demonstrate the potential of monoterpenes overproduction in yeast, which can serve as a generally applicable platform for overproduction of other monoterpenes.

Iridoid Sex Pheromone Biosynthesis in Aphids Mimics Iridoid-Producing Plants.

Biosynthesis of (1R,4aS,7S,7aR)-nepetalactol (1) and (4aS,7S,7aR)-nepetalactone (2) in plants involves iridoid synthase (ISY), an atypical reductive cyclase that catalyses the reduction of 8-oxogeranial into the reactive enol of (S)-8-oxocitronellal, and cyclization of this enol intermediate, either non-enzymatically or by a nepetalactol-related short chain dehydrogenase enzyme (NEPS) that yields the nepetalactols. In this study, we investigated the biosynthesis in vivo of 1 and 2 in the pea aphid, Acyrthosiphon pisum, using a library of isotopically-labelled monoterpenoids as molecular probes. Topical application of deuterium-labelled probes synthesized from geraniol and nerol resulted in production of 2 H4 -lactol 1 and 2 H4 -lactone 2. However, deuterium incorporation was not evident using labelled probes synthesized from (S)-citronellol. These results suggest that iridoid biosynthesis in animals, specifically aphids, may follow a broadly similar route to that characterised for plants.

BHLH IRIDOID SYNTHESIS 3 is a member of a bHLH gene cluster regulating terpenoid indole alkaloid biosynthesis in Catharanthus roseus.

Basic helix‐loop‐helix (bHLH) transcription factors (TFs) are key regulators of plant specialized metabolites, including terpenoid indole alkaloids (TIAs) in Catharanthus roseus. Two previously characterized subgroup‐IVa bHLH TFs, BIS1 (bHLH Iridoid Synthesis 1) and BIS2 regulate iridoid biosynthesis in the TIA pathway. We reanalyzed the recently updated C. roseus genome sequence and discovered that BIS1 and BIS2 are clustered on the same genomic scaffold with a previously uncharacterized bHLH gene, designated as BIS3. Only a few bHLH gene clusters have been studied to date. Comparative analysis of 49 genome sequences from different plant lineages revealed the presence of analogous bHLH clusters in core angiosperms, including the medicinal plants Calotropis gigantea (giant milkweed) and Gelsemium sempervirens (yellow jessamine), but not in the analyzed basal angiosperm and lower plants. Similar to the iridoid pathway genes, BIS3 is highly expressed in roots and induced by methyl jasmonate. BIS3 activates the promoters of iridoid branch genes, geraniol synthase (GES), geraniol 10‐hydroxylase (G10H), 8‐hydroxygeraniol oxidoreductase (8HGO), iridoid synthase (IS), 7‐deoxyloganetic acid glucosyl transferase (7‐DLGT), and 7‐deoxyloganic acid hydroxylase (7DLH), but not iridoid oxidase (IO). Transactivation of the promoters was abolished when BIS3 is converted to a dominant repressor by fusing with the ERF‐associated amphiphilic repression (EAR) sequence. In addition, BIS3 acts synergistically with BIS1 and BIS2 to activate the G10H promoter in tobacco cells. Mutation of the known bHLH TF binding motif, G‐box (CACGTG) in the G10H promoter significantly reduced but did not abolish the transactivation by BIS3. Promoter deletion analysis of G10H suggests that the sequences adjacent to the G‐box are also involved in the regulation by BIS3. Overexpression of BIS3 in C. roseus flower petals significantly upregulated the expression of iridoid biosynthetic genes and increased loganic acid accumulation. BIS2 expression was significantly induced by BIS3 although BIS3 did not directly activate the BIS2 promoter. Our results advance our understanding of the regulation of plant specialized metabolites by bHLH TF clusters.

Characterization of Camptotheca acuminata 10-hydroxygeraniol oxidoreductase and iridoid synthase and their application in biological preparation of nepetalactol in Escherichia coli featuring NADP+ - NADPH cofactors recycling

Nepetalactol, an iridoid with four chiral carbons, is a crucial component of aphid sex pheromones that have been employed with great success to control the insect-related diseases. Despite of agricultural usage as end products, iridoids are fundamental biosynthetic intermediates for pharmaceutically important monoterpenoid indole alkaloids such as camptothecin (CAM) and vinca alkaloids. Herein we characterized 10-hydroxygeraniol oxidoreductase (10HGO) and iridoid synthase (IS) from Camptotheca acuminata, a CAM-producing plant, and reported their application in biological preparation of nepetalactol. Ca10HGO and CaIS were respectively cloned from C. acuminata, overexpressed in Escherichia coli, and purified to homogeneity. Ca10HGO catalyzes the oxidation of 10-hydroxygeraniol into 10-oxogeranial, in which NADP+ was reduced to NADPH. CaIS catalyzes nepetalactol formation from 10-oxogeranial using NADPH cofactor. The net outcome of the two reactions generate nepetalactol from 10-hydroxygeraniol efficiently, indicating NADP+ - NADPH recycling. Ca10HGO and CaIS were co-overexpressed in E. coli under optimized fermentation conditions to prepare cell-based catalysts that catalyze the conversion of 10-hydroxygeraniol into nepetalactol. The present work shows the enzymatic conversion of 10-hydroxygeraniol into nepetalactol involved in CAM biosynthesis. Co-overexpression of Ca10HGO and CaIS in E. coli is an alternative valuable cell-based biotransformation process with regenerating recycling of NADP+ - NADPH cofactors for nepetalactol preparation.

The Progesterone 5β-Reductase/Iridoid Synthase Family: A Catalytic Reservoir for Specialized Metabolism across Land Plants.

Iridoids are plant-derived terpenoids with a rich array of bioactivities. The key step in iridoid skeleton formation is the reduction of 8-oxogeranial by certain members of the progesterone 5β-reductase/iridoid synthase (PRISE) family of short-chain alcohol dehydrogenases. Other members of the PRISE family have previously been implicated in the biosynthesis of the triterpenoid class of cardenolides, which requires the reduction of progesterone. Here, we explore the occurrence and activity of PRISE across major lineages of plants. We observed trace activities toward either 8-oxogeranial or progesterone in all PRISEs, including those from nonseed plants and green algae. Phylogenetic analysis, coupled with enzymatic assays, show that these activities appear to have become specialized in specific angiosperm lineages. This broad analysis of the PRISE family provides insight into how these enzymes evolved in plants and also suggests that iridoid synthase activity is an ancestral trait in all land plants, which might have contributed to the rise of iridoid metabolites.

Engineered mitochondrial production of monoterpenes in Saccharomyces cerevisiae

Monoterpene indole alkaloids (MIAs) from plants encompass a broad class of structurally complex and medicinally valuable natural products. MIAs are biologically derived from the universal precursor strictosidine. Although the strictosidine biosynthetic pathway has been identified and reconstituted, extensive work is required to optimize production of strictosidine and its precursors in yeast. In this study, we engineered a fully integrated and plasmid-free yeast strain with enhanced production of the monoterpene precursor geraniol. The geraniol biosynthetic pathway was targeted to the mitochondria to protect the GPP pool from consumption by the cytosolic ergosterol pathway. The mitochondrial geraniol producer showed a 6-fold increase in geraniol production compared to cytosolic producing strains. We further engineered the monoterpene-producing strain to synthesize the next intermediates in the strictosidine pathway: 8-hydroxygeraniol and nepetalactol. Integration of geraniol hydroxylase (G8H) from Catharanthus roseus led to essentially quantitative conversion of geraniol to 8-hydroxygeraniol at a titer of 227 mg/L in a fed-batch fermentation. Further introduction of geraniol oxidoreductase (GOR) and iridoid synthase (ISY) from C. roseus and tuning of the relative expression levels resulted in the first de novo nepetalactol production. The strategies developed in this work can facilitate future strain engineering for yeast production of later intermediates in the strictosidine biosynthetic pathway.

Molecular cloning and expression analysis of iridoid synthase genes from Rehmannia glutinosa

Iridoid synthase( IS),the key enzyme in the natural biosynthesis of vegetal iridoids,catalyzes the irreversible cyclization of 10-oxogeranial to epi-iridodial. In this study,we screened the Rehmannia glutinosa transcriptome data by BLASTn with Catharanthus roseus CrIS cDNA,and found four c DNA fragments with length of 1 527,1 743,1 425,1 718 bp,named RgIS1,RgIS2,RgIS3 and RgIS4,respectively. Bioinformatics analysis revealed that the four iridoid synthase genes encoding proteins with 389-392 amino acid residues,protein molecular weights were between 44. 30-44. 74 k Da,and theoretical isoelectric points were between 5. 30 and 5. 87. Subcellular localization predictions showed that the four iridoid synthase were distributed in the cytoplasm. Structure analysis revealed that R. glutinosa iridoid synthases contain six conserved short-chain dehydrogenase/reductase( SDR) motifs,and their 3 D models were composed typical dinucleotide-binding " Rossmann" folds covered by helical C-terminal extensions. Using the amino acid sequences of four R. glutinosa iridoid synthases,phylogenetic analysis was performed,the result indicated that RgIS3,CrIS and Olea europaea OeIS were grouped together,the other R. glutinosa iridoid synthases and fifteen proteins in other plants had close relationship. Real-time fluorescent quantitative PCR revealed that RgIS1 and RgIS3 highly expressed in unfold leaves,however,RgIS2 and RgIS4 highly expressed in stems and tuberous roots,respectively. RgIS3 showed higher expression levels in non-radial striations( nRS) of the two cultivars,and RgIS1 and RgIS2 had higher expression levels in nRS of QH,while RgIS4 had less expression levels in nRS of QH1. RgIS1,RgIS2 and RgIS3 were up-regulated by Me JA treatment,although the time and degree of response differed. Our findings are helpful to reveal molecular function of R. glutinosa iridoid synthases and provide a clue for studing the molecular mechanism of iridoid biosynthesis.

Synthetic secrets of catnip compound revealed

Nepetalactone is a cyclic monoterpene best known for instigating the euphoric frenzy that cats and other felines experience when they smell catnip. Now, researchers have discovered that catnip and other plants in the Nepeta genus cyclize the molecule via an unusual two-step pathway (Nat. Chem. Biol. 2018, DOI: 10.1038/s41589-018-0185-2). The discovery may help synthetic biologists engineer microbes to efficiently synthesize closely related molecules that have anticancer and anti-inflammatory properties. Normally, organisms make cyclic monoterpenes with the help of terpene synthase, an enzyme that activates and cyclizes a precursor, geranyl diphosphate, through a cationic intermediate. But nepetalactone is synthesized a little differently: a precursor, 8-oxogeranial, undergoes a reduction before cyclizing. In 2012, Sarah O’Connor, a biochemist at the John Innes Center, discovered iridoid synthase, the enzyme she credited with doing the job. She and her colleagues now report that the pathway is odder than t...

Uncoupled activation and cyclization in catmint reductive terpenoid biosynthesis.

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, catalyzes the activation of its substrate 8-oxogeranial into a reactive enol intermediate, but does not catalyze the subsequent cyclization into nepetalactol. This discovery led us to identify a class of nepetalactol-related short-chain dehydrogenase enzymes (NEPS) from catmint (Nepeta mussinii) that capture this reactive intermediate and catalyze the stereoselective cyclisation into distinct nepetalactol stereoisomers. Subsequent oxidation of nepetalactols by NEPS1 provides nepetalactones, metabolites that are well known for both insect-repellent activity and euphoric effects in cats. Structural characterization of the NEPS3 cyclase reveals that it binds to NAD+ yet does not utilize it chemically for a non-oxidoreductive formal [4 + 2] cyclization. These discoveries will complement metabolic reconstructions of iridoid and monoterpene indole alkaloid biosynthesis.

A multisubstrate reductase from Plantago major: structure-function in the short chain reductase superfamily

The short chain dehydrogenase/reductase superfamily (SDR) is a large family of NAD(P)H-dependent enzymes found in all kingdoms of life. SDRs are particularly well-represented in plants, playing diverse roles in both primary and secondary metabolism. In addition, some plant SDRs are also able to catalyse a reductive cyclisation reaction critical for the biosynthesis of the iridoid backbone that contains a fused 5 and 6-membered ring scaffold. Mining the EST database of Plantago major, a medicinal plant that makes iridoids, we identified a putative 5β-progesterone reductase gene, PmMOR (P. major multisubstrate oxido-reductase), that is 60% identical to the iridoid synthase gene from Catharanthus roseus. The PmMOR protein was recombinantly expressed and its enzymatic activity assayed against three putative substrates, 8-oxogeranial, citral and progesterone. The enzyme demonstrated promiscuous enzymatic activity and was able to not only reduce progesterone and citral, but also to catalyse the reductive cyclisation of 8-oxogeranial. The crystal structures of PmMOR wild type and PmMOR mutants in complex with NADP+ or NAD+ and either 8-oxogeranial, citral or progesterone help to reveal the substrate specificity determinants and catalytic machinery of the protein. Site-directed mutagenesis studies were performed and provide a foundation for understanding the promiscuous activity of the enzyme.

PRISEs (progesterone 5β-reductase and/or iridoid synthase-like 1,4-enone reductases): Catalytic and substrate promiscuity allows for realization of multiple pathways in plant metabolism

PRISEs (progesterone 5β-reductase and/or iridoid synthase-like 1,4-enone reductases) are involved in cardenolide and iridoid biosynthesis. We here investigated a PRISE (rAtSt5βR) from Arabidopsis thaliana, a plant producing neither cardenolides nor iridoids. The structure of rAtSt5βR was elucidated with X-ray crystallography and compared to the known structures of PRISEs from Catharanthus roseus (rCrISY) and Digitalis lanata (rDlP5βR). The three enzymes show a high degree of sequence and structure conservation in the active site. Amino acids previously considered to allow discrimination between progesterone 5β-reductase and iridoid synthase were interchanged among rAtSt5βR, rCrISY and rDlP5βR applying site-directed mutagenesis. Structural homologous substitutions had different effects, and changes in progesterone 5β-reductase and iridoid synthase activity were not correlated in all cases. Our results help to explain fortuitous emergence of metabolic pathways and product accumulation. The fact that PRISEs are found ubiquitously in spermatophytes insinuates that PRISEs might have a more general function in plant metabolism such as, for example, the detoxification of reactive carbonyl species.

Dynamics of loops at the substrate entry channel determine the specificity of iridoid synthases.

Iridoid synthases belong to the family of short-chain dehydrogenase/reductase involved in the biosynthesis of iridoids. Despite having high sequence and structural homology with progesterone 5β-reductase, these enzymes exhibit differential substrate specificities. Previously, two loops, L1 and L2 at substrate-binding pocket, were suggested to be involved in generating substrate specificity. However, the structural basis of specificity determinants was elusive. Here, combining sequence and structural analysis, site-directed mutagenesis, and molecular dynamics simulations, we have shown that iridoid synthase contains two channels for substrate entry whose geometries are altered by L1-L2 dynamics, primarily orchestrated by interactions of residues Glu161 and Gly162 of L1 and Asn358 of L2. A complex interplay of these interactions confer the substrate specificity to the enzyme.

Trichome-specific and developmentally regulated biosynthesis of nepetalactones in leaves of cultivated Nepeta rtanjensis plants

Nepetalactones are accumulated as the main secondary metabolites in the majority of Nepeta species; however, their biosynthetic pathway remains to a great extent unknown, as well as their relationship to the overall physiology of these plants. Nepetalactones most certainly arise through general iridoid biosynthetic pathway leading to nepetalactol, a common precursor of all plant-derived iridoids that is formed in a reaction catalysed by iridoid synthase (IS, EC 1.3.1.99). In this paper, we aimed to localize the sites of nepetalactone biosynthesis and accumulation by comparatively analysing the nepetalactones content and the expression of putative iridoid synthase (NrIS) in leaves of Nepeta rtanjensis Diklić & Milojević, a trans,cis-nepetalactone and dehydronepetalactone rich species. Another aim was to give an insight into the developmental regulation of nepetalactone biosynthesis in leaves of cultivated N. rtanjensis plants. Knowledge on the spatial-, and developmental- regulation of nepetalactone metabolism could greatly benefit potential growers by enabling them to adequately plan and manage plant cultivation, harvesting, and handling and thus increase yield of bioactive compounds.The expression profile of putative NrIS1 is highly correlated with trans,cis-nepetalactone and dehydronepetalactone content/ratio in leaves of different developmental stages. The results strongly indicate that developmental regulation of nepetalactone biosynthesis in leaves resides largely at the level of gene expression, and suggest an important role of NrIS1. Young leaves exhibited the most intensive nepetalactone biosynthesis and accumulation, but also its conversion to dehydronepetalactone. Thus, the pattern of NL accumulation in leaves can be explained by both the rate of its biosynthesis and metabolic degradation.The gene expression patterns of NrIS1 and NrIS2 transcripts in trichomes and abraded leaves revealed trichomes as the main site of their activity. UHPLC/qqqMS profiling of nepetalactones in trichomes and abraded leaves, complemented with Raman spectroscopy and dichloromethane dipping experiment, supported glandular trichome-specific localization of nepetalactone biosynthesis and accumulation.