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

Abstract

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.

The scaffold-forming steps of plant alkaloid biosynthesis

“…The mechanism of NCS (Scheme 24C) has been elucidated through structural 182,183 and computational analyses. 147 Dopamine binds in the enzyme active site prior to 4-HPAA, with the catechol 3-OH H-bonding to the Lys-122 residue deep in the active site and the residues Glu-110 and Asp-141 interact with the dopamine amine group.…”

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

“…These observations are further corroborated by the recent report on four iridoid synthases, NcISY1, NcISY2, NmISY1, and NmISY2, from N. cataria and N. mussinii, respectively. Although all the four of them possess identical or similar residues at 161st (Asp), 162nd (Leu/Trp), and 358th (Cys) positions, only one of the homologs (NcISY2 and NmISY2) exhibit robust activity toward 10-oxogeranial [33]. However, in other set of homologs (NcISY1 and NmISY1), L1 is shorter by one residue which could result in altered dynamics of the loop and hence the binding efficiency of the enzymes for 10-oxogeranial.…”

Biocatalytic Strategies towards [4+2] Cycloadditions

“…We have recently identified three enzymes from the catmint Nepeta mussinii which control formation of nepetalactol, precursor to the cat attractant nepetalactone . These enzymes, the nepetalactol‐related short‐chain‐dehydrogenase/reductases (NEPS), appear to be specific to the Nepeta genus and are putative [4+2] cyclases.…”

“…We have recently identified three enzymes from the catmint Nepeta mussinii which control formation of nepetalactol, precursor to the cat attractant nepetalactone. [36,39] These enzymes, the nepetalactol-related short-chain-dehydrogenase/reductases (NEPS), appeart ob es pecific to the Nepeta genus and are putative [4+ +2] cyclases. The activities of these NAD-dependent enzymes are revealed in cascade reactions with 8-oxogeranial 9,I SY and appropriate cofactors.…”

“…Iridoid monoterpene biosynthesis in plants starts with the reductive cyclization of 8‐oxogeranial 9 to nepetalactol (Scheme B). Through an accumulation of evidence, it has become apparent that the first enzyme in this process, ISY, catalyses the stereoselective 1,4‐reduction of 8‐oxogeranial 9 , but not subsequent cyclisation.…”