Fig. 2: Ancestral sequence reconstruction and activity profile of tetrapods’ FMOs.

a Collapsed phylogeny of FMOs from jawed vertebrates with the targeted ancestors at the nodes: (1) tAncFMO1–5, (2) tAncFMO5, (3) tAncFMO1–4, and (4) tAncFMO1–3 (yellow circles). Clades are colored based on the activity of the mammalian ancestor: S/N oxidation (blue), BV oxidation (orange). No experimental confirmation for FMO4 activity is available. The depicted silhouettes represent the ancestral tetrapod (by Dmitry Bogdanov (vectorized by T. Michael Keesey) under CC BY-SA 3.0) and actinopterygii (ray-finned fish). The scale bar represents the substitutions per site. b Steady-state kinetics for tAncFMO1–4 toward NADPH (red line, K_M = 15.7 ± 1.8 μM) and NADH (blue line, K_M = 90 ± 17.6 μM). Methyl-p-tolyl sulfide (MPTS) was used as substrate. The observed rates were obtained by following the absorbance change at 340 nm. Data are presented as mean values and error bars correspond to SD (n = 3 independent experiments). Source data are provided as a Source Data file. c Conversion assays with the three sets of substrates to test the S/N and BV oxidation activities. Ancestral FMOs from mammals (mAncFMO^18,19) were included in the analysis. Bars represent mean conversion values and dots the values for each experiment (n = 2 independent experiments). S/N oxidations are shown in blue whereas BV oxidations are in orange. Source data are provided in Supplementary Table 4.