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.

The stereochemical variation of the nepetalactones is an intriguing aspect of this biosynthetic pathway. Four nepetalactone stereoisomers with differing stereochemistry at the 4a and 7a positions are observed at different ratios in various Nepeta species (referred to as cis-cis; cis-trans; trans-cis; and trans-trans; Figure 1B) (32)(33)(34). The ratio of the stereoisomers appears to play a role in the plants' ability to deter insects (16,35). However, it is not known how the stereochemistry of these centers is set during the biosynthesis of these compounds. The stereochemistry of the 4a and 7a carbons are established during the cyclization of 8-oxogeranial to nepetalactol. Iridoid synthase (ISY), the enzyme that catalyzes this cyclization reaction to nepetalactol from the starting substrate 8-oxogeranial, could therefore play a role in this stereochemical variation. Alternatively, downstream isomerase enzymes could be responsible for this 6 stereochemical variation. As a first step to understanding the nepetalactone pathway, we set out to discover and characterize the ISYs from Nepeta ( Figure 1B).
In this study, we report the identification of two Nepeta species, N. cataria and N. mussinii, that produce different profiles of nepetalactone diastereomers in leaf tissue: N. cataria produced primarily the cis-trans isomer, while N. mussinii produced primarily the trans-cis isomer ( Figure 1B). Leaf tissue from both species was subjected to a transcriptomic analysis, to provide a resource for understanding the molecular basis for the biosynthesis of these compounds. Candidate ISY enzymes were identified by searching for C. roseus ISY homologs in the transcriptomes. These genes were then cloned, heterologously expressed and functionally characterized by an in vitro assay.
Assays of these Nepeta ISY (NISY) enzymes with 8-oxogeranial revealed that both Nepeta species each harbored two ISY homologs, one of which was substantially more active than the other. These enzymes produced product profiles that were identical to the product profile of the previously reported ISY from Catharanthus roseus, an enzyme known to produce the cis-trans stereoisomer. These results strongly suggest that the variable stereochemistry of the nepetalactone diastereomers is not established during the cyclization reaction catalyzed by ISY, but that a downstream epimerization step must occur. Notably, the Nepeta ISY homologs show surprisingly low sequence identity, as well as changes in the active site residues, to previously characterized ISY enzymes.
This discovery highlights the plasticity of genes and encoded enzymes responsible for the synthesis of the iridoid scaffold.

Selection of plant material
Different species and cultivars of Nepeta produce different profiles of nepetalactone diastereomers (16,33). We obtained local N. cataria and N. mussinii plants and screened the leaves of these plants by GC-MS for production of nepetalactone diastereomers (Figure 2). We found that N. mussinii plants produced primarily the trans-cis (4aS,7S,7aS) lactone, while N. cataria produced primarily the cistrans (4aS,7S,7aR) isomer as evidenced by co-elution with authentic standards on a GC-MS (Figure 2). Both N. cataria and N. mussinii have been reported to produce additional nepetalactone isomers, but the nepetalactone profile likely varies widely from cultivar to cultivar. A third Nepeta species, N. racemosa, was also screened (Figure 2).
This species showed a product profile identical to N. cataria and was not investigated further.

Cloning, heterologous expression and biochemical assay of ISY from Nepeta
Transcriptomes were obtained for leaf tissue of N. mussinii and N. cataria. ISY homologs, which are short chain dehydrogenases, are typically annotated as progesterone 5-b -reductase, the closest known homolog to this enzyme. Two homologs of ISY, from C. roseus (30) and Olea europaea (olive) (36), that have been functionally characterized were used to search the Nepeta transcriptomes for ISY homologs using BLAST. Both Nepeta species each contained two distinct homologs of ISY, which were named family 1 and family 2 (amino acid sequence identity between N. cataria family 1 (NcISY1) and N. cataria family 2 (NcISY2) was 80%; between N. mussinii family 1 (NmISY1) and family 2 (NmISY2) was 79%). Nepeta homologs exhibited amino acid sequence identities to the ISY from C. roseus ranging from 53-58% (Figure 3). All of these proteins are members of the short chain dehydrogenase SDR75U family (http://sdr-enzymes.scilifelab.se/). The Nepeta ISY enzymes were heterologously expressed in E. coli for biochemical characterization, though despite substantial optimization, these enzymes could not be purified to complete homogeneity (Figure 4). The enzymes were incubated with the ISY substrate 8-oxogeranial and NADPH according to previously reported ISY assay conditions, and product formation was monitored by GC-MS (30,37). If the stereochemistry of the 4a and 7a carbons is set by the ISY catalyzed cyclization, then it would be expected that at least one of the N. mussinii ISY enzymes would produce large amounts of trans-cis product. Authentic standards of cis-trans-nepetalactol and iridodials as well as the trans-cis-iridodials (the trans-cis-nepatalactol is not stable (38) could be resolved on the GC-MS chromatogram ( Figure 5A).  The steady-state kinetic constants for NmISY2 were obtained, using an assay based on consumption of NADPH (for 8-oxogeranial, K m = 7.3 ± 0.7 μM, k cat = 0.60 ± 0.02 s -1 ; for NADPH, K m = 26.2 ± 5.1, k cat = 0.84 ± 0.06, all data mean±se.) (Figure 6).
Notably, the availability of the authentic trans-cis-iridodial standard demonstrated that small amounts of the trans-cis isomer were present in all of the enzymatically catalyzed reactions ( Figure 5A). However, due to the low levels of the trans-cis isomer observed in these assays, it seems unlikely that NcISY2 and NmISY2 provide a direct source of this diastereomer.

Sequence of Nepeta ISY
Structural and mechanistic studies of ISY from C. roseus have revealed that the key active site residue is Tyr178 (37,39). This residue is also conserved in all ISY homologs identified from Nepeta (Figure 3). A lysine residue in the active site, Lys146, has been shown to play a catalytic role in progesterone 5-b -reductase, the short chain dehydrogenase most closely related to ISY (40). While this lysine residue is conserved in ISY (C. roseus), mutational analysis demonstrated that this residue does not play an essential role in catalysis (37,39). Nevertheless, along with tyrosine, this lysine forms part of the core conserved residues of the short chain dehydrogenase protein family, of which ISY is a member. While family 1 NISY contained this lysine residue, this residue was replaced with a phenylalanine in members of the more catalytically active family 2.
Moreover, previous work suggests that Gly150 of ISY (C. roseus) allows conformational flexibility of the active site, allowing the enzyme to assume both open and closed forms (37). This residue is mutated to an alanine in family 2 NISY, a mutation that has been shown to lock the active site in an open conformation (37). However, these sequence differences do not appear to alter either the catalytic efficiency or the product profile of these enzymes. This highlights the plasticity of the ISY enzyme responsible for the synthesis of the iridoid scaffold.

Discussion
Members of the Nepeta genus produce nepetalactones, which are iridoid-type monoterpenes that impact plant-insect interactions. The stereochemical variation found among the nepetalactones has a profound influence on the resulting biological activities.
However, the biosynthetic pathway of the nepetalactones at the onset of this work was unknown.
Here, we report the transcriptomes of leaves from two Nepeta plants, N. cataria Surprisingly, the Nepeta ISY enzymes assayed gave identical product profiles ( Figure 5), despite the fact that the stereochemistry of the iridoid scaffolds of these two species are different (Figure 2). The product profile of the Nepeta enzymes was also very similar to the products produced by the previously reported ISY from C. roseus, a plant that produces iridoids exclusively from the cis-trans nepetalactol isomer.
This discovery highlights that the Nepeta ISY enzymes, along with the amino acid sequence differences observed in these enzymes, are not responsible for setting the stereochemistry at the 4a and 7a carbons. Downstream enzymes that catalyze isomerization of nepetalactol or nepetalactone could be required to yield the stereochemical variation that is observed in the Nepeta nepetalactones. The transcriptomic data reported here will facilitate the identification of these downstream enzymes and lead to a greater understanding of the stereochemical variation of these important iridoids produced in Nepeta.

Plant material
Nepeta mussinii and N. cataria were obtained from Herbal Haven, Coldhams

Compounds
Syntheses and isolation of key compounds are outlined below. All of the compounds have been described previously. 1

Cloning and protein expression
cDNA was prepared from leaf RNA using Invitrogen SuperScript III First-Strand Synthesis System kit following the manufacturer's instructions. Primers were designed based on the ISY transcript sequences and with 5'-overhangs for cloning ( Table 2).
These were used to PCR amplify ISY genes from the cDNA and clone directly into a pOPINF expression vector using an InFusion HD cloning kit. The pOPINF vector encodes an N-terminal His-tag. The gene sequences were verified by Sanger sequencing. Two ISY candidates from N. mussinii were obtained (NmISY1 and NmISY2). Sequencing revealed that three ISY candidates were cloned from N. cataria.
Two were cloned from with same primer pair (NcISY1) and had 97% amino acid identity, and thus, only one was investigated further. The two ISY candidates from N.

Kinetic measurement conditions
Kinetics of NADPH consumption were determined spectrophotometrically on a PerkinElmer Lambda 35 instrument at a wavelength of 340 nm in cuvettes with 1 cm path length. Reactions contained 200 mM MOPS buffer pH 7.0, 100 mM sodium chloride and 5 nM NmISY2, in a total volume of 800 μL. 8-oxogeranial was added from a stock solution in inhibitor free THF. A THF co-solvent concentration of 1-1.4% was maintained in the assay to ensure substrate solubility. For determination of 8oxogeranial parameters, 75 μM NADPH was used. For determination of NADPH kinetic parameters, 50 μM 8-oxogeranial was used. Cuvettes were equilibrated to 25 °C before the reaction was initiated by addition of enzyme. Absorbance values were recorded at a rate of 1 Hz. The R software environment was used to fit linear initial rates over 2-5 minutes of the enzyme reaction. Background NADPH consumption was subtracted from initial rates. The Michaelis-Menten equation was fit to data points in R by the nls function to obtain kinetic data. The enzyme NmISY2 appeared to lose activity during storage at -20 °C.

GC-MS method
Samples were injected in split mode (2 μL, split ratio 20:1) at an inlet temperature of 220 °C on a Hewlett Packard 6890 GC-MS equipped with a 5973 mass selective detector (MSD), and an Agilent 7683B series injector and autosampler. Separation was performed on a Zebron ZB5-HT-INFERNO column (5% phenyl methyl siloxane; length: 35 m; diameter: 250 μm) with guard column. Helium was used as mobile phase at a constant flow rate of 1.2 mL/minute and average velocity 37 cm/s. After 5 minutes at 80 °C, the column temperature was increased to 110 °C at a rate of 2.5 K/min, then to 280 °C at 120 K/min, and kept at 280 °C for another 4 minutes. A solvent delay of 5 minutes was allowed before collecting MS spectra at a fragmentation energy of 70 eV.