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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 1. MccB catalyzes C-terminal O-AMPylation of non-native substrates.

Figure 1.

(A) MccB enzymes are members of the E1-like/ThiF superfamily, which have in common the formation of a C-terminal peptidyl-O-AMP intermediate. (B) Members of the E1-like superfamily that act on C-termini generally recognize ubiquitin-like proteins that terminate in Gly as their substrates; the MccB family is an exception as it acts on short peptides (MccAs) that terminate in Asn. (C) The reaction catalyzed by MccB in its native context. (D) The C-terminal residue of E. coli MccA was varied to the 19 non-native amino acids and MccB’s ability to catalyze C-terminal O-AMPylation was assayed using a colorimetric assay for pyrophosphate. (E) MccA-N7A, N7G, N7S, and N7T stimulate MccB-catalyzed pyrophosphate formation (left column), while only wt MccA supports formation of a stably AMPylated product detectable by HPLC-MS (right column). (F) Steady-state kinetic parameters for MccB-catalyzed pyrophosphate release when wt MccA or MccA variants are used as substrates. Kinetic parameters are given as value ± standard error.