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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 4a and 7a positions e which are generated during the iridoid synthase reaction e
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.