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. Author manuscript; available in PMC: 2026 Jul 18.
Published in final edited form as: Nat Chem. 2025 Jul 18;17(9):1371–1382. doi: 10.1038/s41557-025-01871-3

Figure 2. MccB catalyzes formation of a peptidyl-O-AMP intermediate that can react with exogenous nucleophiles.

Figure 2.

(A) MccA-O-AMP undergoes reaction with its C-terminal Asn side chain to form a succinimide intermediate and subsequently a stably N-AMPylated product that can be detect by LC-MS (top). In contrast, MccA-N7G-O-AMP is expected to undergo hydrolysis on a timescale incompatible with LC-MS detection. (B) LC-MS analysis of MccB reactions with wild-type MccA (left) or MccA-N7G (right) as a substrate. (C) Hypothesized MccA-N7G-O-AMP reactivity with exogenous nucleophiles. (D) MccB-catalyzed hydrazine modification of MccA-N7G. ESI-MS spectra of unmodified MccA-N7G (left) and MccA-N7G (250 μM) incubated with MccB (5 μM), ATP (5 mM), and hydrazine (150 mM) reveal that MccA-N7G-O-AMP can react with exogenous nucleophiles. (E) Radial heatmaps show the percent conversion of MccA to nucleophile-modified MccA for a panel of alkoxyamine, hydrazine, amine, and thiol nucleophiles. Percent conversion was calculated using peak areas from reactant and product extracted ion chromatograms.