Identification of AmISY. a, phylogenetic tree of iridoid synthase homologues in A. majus (Am), Olea europaea (Oe), C. roseus (Cr), Nepeta cataria (Nc), and N. mussinii (Nm). The neighbor joining tree was built from a MuscleWS alignment using the BLOSUM62 similarity matrix in Jalview 2.10.1 (24). Numbers next to the nodes indicate evolutionary distances. Proteins with proven iridoid synthase activity are highlighted in bold font. One of the A. majus homologues (AmISY or Am18679) groups closely with the iridoid synthases from O. europaea and C. roseus. b, SDS-PAGE of nickel affinity- and gel filtration chromatography–purified AmISY. c, the 8-oxogeranial–dependent NADPH consumption of AmISY showed catalytic parameters close to those of CrISY at a fixed NADPH concentration of 50 μm (AmISY: kcat = 0.72 ± 0.02 s−1, Km = 1.1 ± 0.1 μm; CrISY: kcat = 1.6 ± 0.1 s−1, Km = 4.5 ± 0.2 μm). Values are given as the mean ± S.D. of two independent measurements with different batches of protein. d, qRT-PCR shows tissue-dependent expression of ISY homologues in A. majus. Abundance of the Am29566 transcript was too low for quantification in all tissues. Expression values are given as the mean ± S.D. (four reactions). Each gene was separately normalized to the tissue with the highest expression level. Two replicates each were analyzed for two independent samples of cDNA.