Functional and Mechanistic Investigation of a Distinct DUF6895 Family Epimerase Involved in Lasso Peptide Modification

The domain of unknown function 6895 (DUF6895) constitutes a poorly characterized protein family. Its functional obscurity positions this family of proteins as high-value targets to unlock cryptic enzymatic activities and molecular mechanisms. Here, we identified a DUF6895-encoding gene within a lasso peptide biosynthetic gene cluster. The DUF6895 protein ShpE functions as a distinct epimerase that inverts the configuration of phenylalanine in the linear precursor peptide, a modification essential for the subsequent tryptophan dihydroxylation. Structural and mechanistic analyses demonstrated that ShpE provides a hydrophobic cavity to accommodate phenylalanine and employs acid–base chemistry to facilitate reversible epimerization via dual His/Glu catalytic dyads. These dyads are evolutionarily conserved across DUF6895 proteins, suggesting the majority of these family members function as epimerases through a common molecular mechanism. Our work on ShpE expands the enzymatic repertoire for epimerization in ribosomally synthesized and post-translationally modified peptides (RiPPs), providing crucial insights into the function and mechanism of the neglected DUF6895 proteins.