Uncoupled activation and cyclisation in catmint reductive terpenoid biosynthesis

Abstract

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 nonoxidoreductive formal [4+2] cyclisation. These discoveries will complement metabolic reconstructions of iridoid and monoterpene indole alkaloid biosynthesis. Main paperNepetalactones 1 are volatile natural products produced by plants of the genus Nepeta, notably catmint (Nepeta mussinii syn racemosa) and catnip (N. cataria) (Fig. 1a) 1,2 . These compounds are responsible for the stimulatory effects these plants have on cats [3][4][5] . Moreover, certain insects use nepetalactones as sex pheromones, so production of these compounds by the plant also impacts interactions with insects 6 . Notably, the bridgehead stereocentres (carbons 4a and 7a) vary between 2 and within 2,4,7 Nepeta species. N. mussinii individuals, for example, produce different ratios of cis-trans 1a, cis-cis 1b and trans-cis-nepetalactone 1c 7 . Variation in stereoisomer ratio may influence the repellence of insect herbivores 8,9 . While the ratio of stereoisomers may be responsible for important biological effects, the mechanism of stereocontrol in nepetalactone biosynthesis is not known.Nepetalactones are iridoids, non-canonical monoterpenoids containing a cyclopentanopyran ring. Canonical cyclic terpenoids (e.g. (-)-limonene) are biosynthesised from linear precursors by terpene synthases (Fig. 1b) 10 . These enzymes activate linear precursors either by loss of pyrophosphate or protonation [10][11][12] . The resulting carbocations generated cyclise rapidly to form an array of cyclic products 13 . Therefore, in canonical terpenoid biosynthesis, activation and cyclisation of precursors are coupled and occur in the same enzyme active site.In plant iridoid biosynthesis, geranyl pyrophosphate is hydrolysed and oxidised into 8-oxogeranial 2 14 . This precursor then undergoes a two-step activation-cyclisation process, analogous to canonical terpene synthesis (Fig. 1c) 15 . Unlike canonical terpene synthesis, however, activation is achieved by reduction, and the intermediate is not a carbocation, but the enol or enolate species 3. Cyclisation of this intermediate yields cis-trans-nepetalactol 4a along with iridodial side products 5 (Fig. 1c).

The evolutionary origins of the cat attractant nepetalactone in catnip

“…The previously observed cis-cis cyclase activity of NmNEPS3 was also obtained here, together with the characterization of two new NEPS3 enzymes from N. cataria (9). As expected, both NcNEPS3A and NcNEPS3B formed cis-cis-nepetalactol (Table 1 and fig.…”

“…(B) Early iridoid biosynthetic pathway in plants: geraniol synthase (GES), geraniol 8-hydroxylase (G8H), 8-hydroxygeraniol oxidoreductase (HGO), and iridoid synthase (ISY). (C) Knowledge of nepetalactone biosynthesis in N. mussinii as reported in Lichman et al (9). The crossed double bond depiction in the ISY product refers to unknown or undefined stereochemistry.…”

“…Genes of interest were identified by blast searching gene models with previously characterized iridoid biosynthesis enzymes from Nepeta (NEPSs and PRISEs) and Catharanthus (GES, G8H, and HGOA) (5,7,9,10). The genomic locations and tissue expression patterns of the genes were used to assess whether the genes were genomically clustered and/or coexpressing with other iridoid biosynthetic genes.…”

“…Instead, ISYs catalyze the reduction of 8OG but do not control the subsequent cyclization. These enzymes yield a reactive intermediate that, without partner enzymes present, spontaneously cyclizes to form a mixture of products including cis-transnepetalactol (8,9).…”

“…In Nepeta mussinii, several enzymes act in combination with ISY to control the formation of nepetalactone stereoisomers (Fig. 1C) (9). Three nepetalactol-related short-chain reductase/dehydrogenases (NEPS1, NEPS2, and NEPS3) were identified.…”