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.