Fig. 4

Probing NosN reaction by using substrate analogues. a NosN methylates two MIA-based thioester (5 and 9) to produce the corresponding indole C4 methylated products (6 and 10). b Extracted ion chromatograms (EICs) of [M+H]⁺  = 450.2 (corresponding to DMIA-Pan, 6) for (i) control reaction with the supernatant of boiled NosN, and (ii) overnight reaction with NosN and 5. c Time-course analysis of the NosN reactions with MIA-SNAC 9 and MIA-Pan 5, showing that 5 is a better substrate of NosN than 9. The reactions were carried out with 100 μM substrates (5 or 9), 1 mM SAM, 1 mM NADPH, 50 µM FldA, 20 µM Fpr and 100 μM NosN. The chemical structures of DMIA (11) and 12 are shown; the latter is a structural mimic of the MIA-based thioester associated with a polypeptide Cys residue, which is not a NosN substrate