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. Author manuscript; available in PMC: 2019 May 14.
Published in final edited form as: Nat Chem Biol. 2018 Dec 10;15(1):71–79. doi: 10.1038/s41589-018-0185-2

Fig. 3. Formation of nepetalactones by NEPS enzymes.

Fig. 3

a, Summary of NEPS enzyme activities described in this figure. b, cis-trans-Nepetalactol dehydrogenase activity of NEPS1. NEPS1 catalyzes the NAD+-dependent dehydrogenation of 4a to 1a. c, cis-cis-Nepetalactol dehydrogenase activity of NEPS1. NEPS1 catalyzes the NAD+-dependent dehydrogenation of 4b to 1b. NEPS1 activities on 4a and 4b are consistent with cascade activities (d), kinetics (Supplementary Table 2) and activities with 6 (Fig. 4). d, Combined activities of ISY and NEPS enzymes. Incubation of 2, CrISY, NEPS and cofactors enables the production of 4a (no NEPS or NEPS2), 1a (NEPS1), 4b (NEPS3) or 1b (NEPS1 and NEPS3). Experiments performed under nearly identical conditions also provided similar results (Supplementary Fig. 12). Observed enzyme activities are consistent with activities observed in Fig. 4b. e, NEPS-catalyzed formation of 4a. Adjusting the NAD+ and/or NEPS concentrations reveals that NEPS1 and NEPS2 can promote the formation of 4a. See Supplementary Fig. 9 for EI spectra, Supplementary Fig. 12 for NEPS cascades with NmISY2 and CrISY, and Supplementary Fig. 13 for further analysis of the NEPS3 cyclization reaction. All experiments were performed independently twice with similar results.