Biosynthesis of the RiPP trojan horse nucleotide antibiotic microcin C is directed by the -formyl of the peptide precursor.

Microcin C7 (McC) is a peptide antibiotic modified by a linkage of the terminal isoAsn amide to AMP a phosphoramidate bond. Post-translational modification on this ribosomally produced heptapeptide precursor is carried out by MccB, which consumes two equivalents of ATP to generate the N-P linkage. We demonstrate that MccB only efficiently processes the precursor heptapeptide that retains the -formylated initiator Met (fMet). Binding studies and kinetic measurements evidence the role of the -formyl moiety. Structural data show that the -formyl peptide binding results in an ordering of residues in the MccB "crossover loop", which dictates specificity in homologous ubiquitin activating enzymes. The -formyl peptide exhibits substrate inhibition, and cannot be displaced from MccB by the desformyl counterpart. Such substrate inhibition may be a strategy to avert unwanted McC buildup and avert toxicity in the cytoplasm of producing organisms.