The bottromycin epimerase BotH defines a group of atypical α/β-hydrolase-fold enzymes

d -amino acids endow peptides with diverse, desirable properties, but the post-translational and site-specific epimerization of l -amino acids into their d -counterparts is rare and chemically challenging. Bottromycins are ribosomally synthesized and post-translationally modified peptides that have overcome this challenge and feature a d -aspartate ( d -Asp), which was proposed to arise spontaneously during biosynthesis. We have identified the highly unusual α/β-hydrolase (ABH) fold enzyme BotH as a peptide epimerase responsible for the post-translational epimerization of l -Asp to d -Asp during bottromycin biosynthesis. The biochemical characterization of BotH combined with the structures of BotH and the BotH–substrate complex allowed us to propose a mechanism for this reaction. Bioinformatic analyses of BotH homologs show that similar ABH enzymes are found in diverse biosynthetic gene clusters. This places BotH as the founding member of a group of atypical ABH enzymes that may be able to epimerize non-Asp stereocenters across different families of secondary metabolites. BotH is an unusual α/β-hydrolase-fold enzyme that catalyzes epimerization of an aspartate residue during bottromycin biosynthesis via a mechanism distinct from other known peptide natural product epimerases.