Fig. 4: Mammalian FMO metabolomics analysis reveals the tryptophan/kynurenine pathway as a target of FMO-2.

a Conserved catalytic residues between CeFMO-2 and mFMO5 (indicated by red arrows). b The level of phenylalanine and tryptophan present in HepG2 cells expressing pDEST control vector, mFMO2, mFMO4, and mFMO5. Black dot = vector control and blue triangle = FMO OE. * represents p < 0.05 by two-tailed unpaired Student’s t-test. For phenylalanine, p-value = 0.0084 (control vs mFMO2 OE), 0.00095 (control vs mFMO4 OE), 0.04 (control vs mFMO5 OE). For tryptophan, p-value = 0.0188 (control vs mFMO2 OE), 0.000918 (control vs mFMO4 OE), 0.098 (control vs mFMO5 OE). Data is represented as mean values +/− SD, n = 3 biological replicates. c, d The reaction rate by concentration for purified CeFMO-2 enzyme toward tryptophan (n = 7 biologically independent replicates) and phenylalanine (n = 2 biologically independent replicates) at 30 °C. Data is represented as mean values +/− SD. e The abundance of N-formylkynurenine based on LC-MS analysis of CeFMO-2 activity toward 100, 250, and 500 μM tryptophan at 30 °C. n = 3 (100 µM and 250 µM Tryptophan) and n = 2 (500 µM Tryptophan) biologically independent replicates. Data is represented as mean values +/− SD. f Comparison of targeted metabolomics data of tryptophan between the wild type (black), FMO-2 OE (blue), and FMO-2 KO (red), normalized to the average of wild type intensity. n = 4 biologically independent replicates and p-value = 0.028 (WT vs FMO-2 OE). * = p < 0.05 using two-tailed unpaired Student’s t-test. g Summary table of Michaelis-Menten parameters for CeFMO-2 cofactor and substrate. h Comparison of targeted metabolomics data of phenylalanine between the wild type (black), FMO-2 OE (blue), and FMO-2 KO (red), normalized to the average of wild type intensity. n = 4 biologically independent replicates and p-value = 0.0097 (WT vs FMO-2 OE). ** = p < 0.01 using two-tailed unpaired Student’s t-test. i Lifespan assay comparing the survival of the wild type, FMO-2 OE, and FMO-2 KO on control and 1 mM formate supplementation conditions. Black circle = wild type, blue diamond = FMO-2 OE, and red triangle = FMO-2 KO. Solid line = EV, dotted line = formate. * denotes significant change in mean lifespan at p < 0.05 using log-rank and # denotes significant interaction between the condition of interest and fmo-2 genotype at p < 0.01 using Cox regression analysis. N.S. = not significant. Statistics are in Supplementary Table 10 and Supplementary Data 5. For box plots in f and h, the median is shown by the center line. The upper boundary of the box represents the 75% interquartile range, while the lower boundary represents the 25% interquartile range.