Fig. 3. In vitro analysis of DarE_pk.

a The EIC and HRMS spectrum corresponding to the tryptic fragment DarA_pk(43-53)-2, which contains an ether-crosslink between Trp49 and Trp51. b The EIC and HRMS spectrum corresponding to the tryptic fragment DarA_pk(43-53)-1 containing the fully modified bicyclic scaffold. c The EIC and HRMS spectrum corresponding to the tryptic fragment DarA_pk(43-53)-3 likely containing a dehydrogenated Trp51 (dhW). Trace i, ii, and iii correspond to the full reaction, the solution of reconstituted DarE_pk, and the negative control with the supernatant of heat-inactived enzyme. d HRMS spectra of the tryptic fragments DarA_pk(43-53)-2 generated in (i) a buffer containing ~80% H₂¹⁸O, which was obtained by ultrafiltration and H₂¹⁸O dilution. The asterisk indicates ¹⁸O incorporation (~50% enrichment) into the resulting peptide. A control experiment (ii) was performed in parallel with H₂O. See Supplementary Fig. 12 for the detailed comparison of the HR-MSMS spectra for the ¹⁸O-labeled vs. unlabeled tryptic fragments DarA_pk(43–53)−2.