Microbial dl-Peptidases Enable Predator Defense and Facilitate Structure Elucidation of Complex Natural Products

Peptidases are indispensable tools in biotechnology and chemical biology. However, the enzyme repertoire for the selective hydrolysis of dl-amide bonds in peptides is small. Here, we describe novel dl-peptidases that mediate complex microbial interactions. These enzymes, Lip3 and Lip7, convert lipopeptides into potent amoebicidal agents via selective dl-peptide bond cleavage. Using structural analyses and mutagenesis, we identified an unusual Ser-Lys-Lys-Tyr catalytic tetrad required for dl-specificity. Despite their high structural similarity, both enzymes show distinct substrate preferences: Lip3 acts primarily as a carboxypeptidase, removing a single C-terminal residue, while Lip7 excises a tripeptide. Although their substrate scopes are broad, they are highly specific with regard to their respective cutting sites. These features make these dl-peptidases powerful tools for elucidating the structure of complex peptide-based natural products, including tensin and WLIP. Overall, this work elucidates the molecular mechanisms of cooperative microbial defense and provides a new enzymatic toolbox for biocatalysis and natural product discovery.