Recycling Upstream Redox Enzymes Expands the Regioselectivity of Cycloaddition in Pseudo-Aspidosperma Alkaloid Biosynthesis

Nature uses cycloaddition reactions to generate complex natural product scaffolds. Dehydrosecodine is a highly reactive biosynthetic intermediate that undergoes cycloaddition to generate several alkaloid scaffolds that are the precursors to pharmacologically important compounds such as vinblastine and ibogaine. Here we report how dehydrosecodine can be subjected to redox chemistry, which in turn allows cycloaddition reactions with alternative regioselectivity. By incubating dehydrosecodine with reductase and oxidase biosynthetic enzymes that act upstream in the pathway, we can access the rare pseudoaspidosperma alkaloids pseudo-tabersonine and pseudo-vincadifformine, both in vitro and by reconstitution in the plant Nicotiana benthamiana from an upstream intermediate. We propose a stepwise mechanism to explain the formation of the pseudo-tabersonine scaffold by structurally characterizing enzyme intermediates and by monitoring the incorporation of deuterium labels. This discovery highlights how plants use redox enzymes to enantioselectively generate new scaffolds from common precursors

Phylogenomic Mining of the Mints Reveals Multiple Mechanisms Contributing to the Evolution of Chemical Diversity in Lamiaceae

The evolution of chemical complexity has been a major driver of plant diversification, with novel compounds serving as key innovations. The species-rich mint family (Lamiaceae) produces an enormous variety of compounds that act as attractants and defense molecules in nature and are used widely by humans as flavor additives, fragrances, and anti-herbivory agents. To elucidate the mechanisms by which such diversity evolved, we combined leaf transcriptome data from 48 Lamiaceae species and four outgroups with a robust phylogeny and chemical analyses of three terpenoid classes (monoterpenes, sesquiterpenes, and iridoids) that share and compete for precursors. Our integrated chemical–genomic–phylogenetic approach revealed that: (1) gene family expansion rather than increased enzyme promiscuity of terpene synthases is correlated with mono- and sesquiterpene diversity; (2) differential expression of core genes within the iridoid biosynthetic pathway is associated with iridoid presence/absence; (3) generally, production of iridoids and canonical monoterpenes appears to be inversely correlated; and (4) iridoid biosynthesis is significantly associated with expression of geraniol synthase, which diverts metabolic flux away from canonical monoterpenes, suggesting that competition for common precursors can be a central control point in specialized metabolism. These results suggest that multiple mechanisms contributed to the evolution of chemodiversity in this economically important family. The mint family (Lamiaceae) includes many culturally and economically important species and collectively exhibits an exceptionally high degree of chemical diversity. Using an integrated chemical-genomic-phylogenetic approach, gene family expansion, altered gene expression of key biosynthetic pathway genes, and flux of precursors were shown to underlie the evolution of chemodiversity observed in this chemically rich clade

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

Nepetalactones 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 4α and 7α 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 4α and 7α 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

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 effect 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

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

Abstract Blueberries (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

T_iboga_2.fq.gz

The files T_iboga_2.fq.gz.001 and T_iboga_2.fq.gz.002 must be joined into a single file before decompressing. Join the files using the following command indicated for your operating system. Linux/Mac OS X: cat T_iboga_2.fq.gz.* > T_iboga_2.fq.gz Windows: copy /b T_iboga_2.fq.gz.001 + T_iboga_2.fq.gz.002 T_iboga_2.fq.g

T_iboga_2.fq.gz

The files T_iboga_2.fq.gz.001 and T_iboga_2.fq.gz.002 must be joined into a single file before decompressing. Join the files using the following command indicated for your operating system. Linux/Mac OS X: cat T_iboga_2.fq.gz.* > T_iboga_2.fq.gz Windows: copy /b T_iboga_2.fq.gz.001 + T_iboga_2.fq.gz.002 T_iboga_2.fq.g

1_T_iboga_1.fq.gz

These are illumina reads. The files T_iboga_1.fq.gz.001 and T_iboga_1.fq.gz.002 must be joined into a single file before decompressing. Join the files using the following command indicated for your operating system. Linux/Mac OS X: cat T_iboga_1.fq.gz.* > T_iboga_1.fq.gz Windows: copy /b T_iboga_1.fq.gz.001 + T_iboga_1.fq.gz.002 T_iboga_1.fq.g

2_T_iboga_1.fq.gz

The files T_iboga_1.fq.gz.001 and T_iboga_1.fq.gz.002 must be joined into a single file before decompressing. Join the files using the following command indicated for your operating system. Linux/Mac OS X: cat T_iboga_1.fq.gz.* > T_iboga_1.fq.gz Windows: copy /b T_iboga_1.fq.gz.001 + T_iboga_1.fq.gz.002 T_iboga_1.fq.g