Fig. 3: TsaM demonstrates a preference for oxygenating the methyl group or benzylic carbon of substrates that contain a polar functional group.

a TsaM oxygenates p-toluenesulfonate (1), 4-methylbenzoate (2), p-aminotoluene (5), and p-nitrotoluene (6). In contrast, substrates that lack a charged functional group at the p-position (p-isopropyltoluene, 7, toluene, 11 and p-chlorotoluene, 13) are not functionalized by TsaM. The reported^43,44,45 native C1 substrate functional groups are indicated in white. b TsaM accepts and oxygenates 3-methylbenzoate (15) but does not oxygenate 2-methylbenzoate (17). This panel reflects previously described results⁴⁸, and, like panel a, also shows activity as a heat map with the native p-position of oxygenation in white. c The AlphaFold^52,53 model of TsaM⁴⁸, which is visualized using PyMOL 2.5.2_93 software, highlights a putative binding site for substrate (dashed circle). d Providing a 4-ethylbenzoate (19) or 4-isopropylbenzoate (25) to TsaM results in production of monooxygenated and desaturated products. The monooxygenated products are 4-(1-hydroxyethyl)benzoate (20) and 4-(2-hydroxy-2-propyl) benzoate (26), respectively. Total turnover numbers (TTN) were also measured for TsaM with a 4-propyl- (28), 4-butyl- (29), and 4-pentylbenzoate (30). These reactions also showed formation of both oxygenated and desaturated products, but the desaturated products were not quantified. TTNs are colored as described in panel a. e As previously described for p-(methoxy)benzoate⁴⁸ (31), TsaM catalyzes oxidative dealkylation chemistry when provided with p-(methylamino)benzoate (33) and p-(methylthio)benzoate (34). f A plot of the amount of H₂O₂ generated in the TsaM-VanB catalyzed reactions reveals a significant increase in H₂O₂ formation when p-nitrotoluene and p-isopropyltoluene are provided as substrates to TsaM-VanB. In this panel ****p < 0.0001 and ns indicates no significant difference from an ordinary one-way ANOVA Tukey analysis. P values from left to right >0.9999, 0.9949, <0.0001, 0.8859, <0.0001, 0.7562, >0.9999, >0.9999, >0.9999, 0.7595, 0.0614, 0.1727, >0.9999, >0.9999, >0.9999, and >0.9999. In all panels, data was measured using n = 3 independent experiments and is presented as the mean value of these measurements. In panels d–f data are presented as mean values ± SD. For panels a–c and f additional details are provided in Supplementary Figs. 12, 15, and 17. Source data are provided as a Source Data file.