M icrocin C (McC) is heptapeptide-adenylate antibiotic produced by Escherichia coli strains carrying the mccABCDEF gene cluster encoding, in addition to the heptapeptide structural gene mccA, enzymes necessary for McC biosynthesis and self-immunity of the producing cell. The heptapeptide facilitates McC transport into susceptible cells, where it is processed releasing a non-hydrolyzable aminoacyl adenylate that inhibits an essential aspartyl-tRNA synthetase. The self-immunity gene mccF encodes a specialized serine-peptidase that cleaves an amide bond connecting the peptidyl or aminoacyl moieties of, respectively, intact and processed McC with the nucleotidyl moiety. Unlike in E. coli, some of mccF orthologs are not expressed as a part of the mcc operon, and exist as single genes. Here, we show that a protein product of one such gene, MccF from Bacillus anthracis (BaMccF), is able to cleave intact and processed McC. Structural analysis conformed this observation. The structures of apo-BaMccF and its AMP-complex revealed a peptidase with specific features that allow MccF to interact with substrates containing nucleotidyl moieties. Sequence analysis and phylogenetic tree reconstruction for the MccF/LD-carboxypeptidase family of proteins show distinct subfamilies in the MccF clade. Several representatives of MccF clade can restore E. coli resistance to McC and other non-hydrolyzable aminoacyl adenylates. Based on our data we propose that members of MccF clade may have similar substrate specificity. Our results suggest that expression of widespread mccF-like genes may be linked to detoxification of aminoacyl adenylates (endogenous or exogenous) and may represent a "stealthy" source of antibiotic resistance.