Structure and substrate recognition of the Bottromycin maturation enzyme BotP

The bottromycins are a family of highly modified peptide natural products displaying potent antimicrobial activity against Gram-positive bacteria including methicillin-resistant Staphyloccoccus aureus. Bottromycins have recently been shown to be ribosomally synthesized and post-translationally modified peptides (RiPPs). Unique amongst RiPPs the precursor peptide BotA contains a C-terminal "follower" sequence, rather than the canonical N- terminal "leader" sequence. We report the structural and biochemical characterization of BotP, a leucyl-aminopeptidase like enzyme from the bottromycin pathway. We demonstrate that BotP is responsible for the removal of the N-terminal methionine from the precursor peptide. Determining the crystal structures of apo BotP and of BotP in complex with Mn²⁺ allowed us to model a BotP/substrate complex and to rationalize substrate recognition. Our data represent the first step towards targeted compound modification to unlock the full antibiotic potential of bottromycin.