Lichman, B. | bioRxiv Science

bioRxiv2018

Uncoupled activation and cyclisation 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, 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 non-oxidoreductive formal [4+2] cyclisation. These discoveries will complement metabolic reconstructions of iridoid and monoterpene indole alkaloid biosynthesis.

biochemistry↗

bioRxiv2017

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

Graphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC=\"FIGDIR/small/179572_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (91K):\norg.highwire.dtl.DTLVardef@cc600dorg.highwire.dtl.DTLVardef@1b6240dorg.highwire.dtl.DTLVardef@f8e1beorg.highwire.dtl.DTLVardef@1acb9dd_HPS_FORMAT_FIGEXP M_FIG Iridoid synthase from Nepeta cateria (catnip) and Nepeta mussinii, have been cloned and characterized.\n\nC_FIG 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 4 and 7 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 4 and 7 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.\n\nHighlightsO_LISpecies within the Nepeta genus (such as catnip) produce nepetalactone iridoids\nC_LIO_LIThe enzymes that produce the iridoid scaffold of nepetalactone were identified from two species of Nepeta\nC_LIO_LIThe iridoid synthase enzymes are not responsible for the stereochemical variation in these iridoids\nC_LI

biochemistry↗