Extended Data Fig. 4: Background conversion of the substrate analogue 8.

Spontaneous transformation of the substrate analogue 8 into the endo 9 and exo 10 adducts occurs almost entirely in the HPLC solvent supplemented with formic acid. 8 (150 µM) was incubated for 0 (labelled “buffer 0 min” in the plot), 10 (buffer 10 min) and 20 min (buffer 20 min) at 25 °C without CghA in the reaction buffer (100 mM potassium phosphate, 100 mM NaCl, pH 8.4) in a total reaction volume of 25 µl. After incubation, the reaction was quenched with 25 µl of MeOH and then centrifuged for removal of debris. Subsequently, the sample was subjected to LC–MS analysis. For the control experiment “buffer 10 min + CghA 10 min”, 8 (150 µM) was incubated for 10 min at 25 °C without CghA in the reaction buffer to allow the spontaneous transformation of 8. Subsequently, 0.1 µM of the wild-type CghA was added to the reaction mixture and the mixture was incubated for another 10 min at 25 °C in a total reaction volume of 25 µl to allow CghA to react on the remaining 8. For other control experiments “CghA 10 min”, “CghA + 10 10 min” and “CghA + 9”, 150 µM of 8, 10 and 9 was added to the reaction mixture, respectively. These mixtures were incubated for 10 min at 25 °C with CghA (0.1 µM wild-type CghA) under the same conditions and analysed as described above.