Fig. 4: TsaM produces a sequentially monooxygenated product when provided with a substrate that lacks a methyl group.

a Incubation of TsaM-VanB with benzenesulfonate (37) or benzoate (38) results in production of 3,4-dihydroxybenzenesulfonate (39) or 3,4-dihydroxybenzoate (40), respectively. Formation of these species can be accomplished either via sequential monooxygenation reactions or a single dioxygenation event. b Using the H₂O₂ shunt reaction, it was determined that incubation of benzenesulfonate with TsaM results in production of a monooxygenated compound, 4-hydroxybenzenesulfonate (41), after a single turnover. Analogous results are obtained when benzoate is included as the substrate of the reaction. c Similarly, using the H₂O₂ shunt reaction, it was shown that 4-hydroxybenzoate (32) and 4-hydroxybenzenesulfonate can be transformed into 3,4-dihydroxybenzenesulfonate and 3,4-dihydroxybenzoate following an additional turnover, respectively. d Inspection of the TsaM-catalyzed reaction when provided with benzenesulfonate or benzoate reveals that first a monooxygenated species (purple and green) is produced and then a dioxygenated (light blue and pink) species is formed. In panel d the data was measured using n = 2 independent experiments. Additional details that pertain to panel d can be found in Supplementary Figs. 36–38. Source data are provided as a Source Data file.