The cis-iridodial and trans-iridodial freely interconverted with cis-trans-nepetalactol, and although CYP76A26 seemed to use the bicyclic nepetalactol as the preferred substrate, the monocyclic cis- and trans-iridodials were also utilized, possibly after spontaneous conversion into nepetalactol (Fig. 4). The interconversion and sequential metabolism of nepetalactol and the iridodials in aqueous solution prevented reliable evaluation of the catalytic parameters with these substrates. Iridotrial was previously proposed as an intermediate of the secologanin pathway. Whereas we never detected iridotrial as an intermediate in the iridodial to 7-deoxyloganetic acid conversion under conditions more likely to capture early reaction products such as low substrate concentrations, short incubations or low temperature, the latter was very efficiently converted into 7-deoxyloganetic acid with a Kmapp of 25 mM and kcat of 5.2 s1 (Supplementary Fig. 3). This suggests that CYP76A26 is a multifunctional P450 enzyme catalysing successive hydroxylation/dehydration steps via a mechanism
similar to that recently described for CYP701A3, which catalyses the conversion of kaurene to kaurenoic acid in the biosynthesis of gibberellins