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. 2022 Feb 22;119(8):e2110293119. doi: 10.1073/pnas.2110293119

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Fig. 4. — Biochemical analysis of Mtb Rv0097–Rv0101 gene cluster. (A) Schematic representation of putative biosynthetic steps for unknown metabolite production by Mtb Rv0097–Rv0101 gene cluster. Mtb harbors bimodular NRPS with seven catalytic domains. (B) Reaction scheme for enzymatic assays of purified Rv0098 and Rv0097 proteins with chemically synthesized C12-SNAC as substrate. (C) MS/MS spectra showing fragmentation products of glycine adduct of 2-dodecenoic-SNAC, 1, detected in the enzymatic assay with Rv0098 purified protein. No product could be detected in control reactions with no protein or no glycine. (D) MS/MS spectra of additional peak of m/z 326.2028 corresponding to isonitrile adduct of 2-dodecanoic-SNAC, 2, observed upon addition of Rv0097 purified protein to the Rv0098 assay conditions. The calculated masses for metabolites 1 and 2 are within 5-ppm mass error tolerance at MS1. All assays in C and D were performed in triplicates with reproducible results each time.