Structural characterization of an S8 protease from Fervidobacterium pennivorans reveals a unique tyrosine-rich β-hairpin.

Keratin is an abundant structural fibrous protein and extremely recalcitrant biopolymer. β-Keratin is the major constituent of feathers, which, due to the widespread poultry industry, has become a major waste product. Biotechnological upcycling of feather waste has gained interest as various bacteria and fungi capable of degrading keratin have been isolated. These microorganisms produce proteases, termed keratinases, responsible for the enzymatic hydrolysis of keratin. The structural properties that confer keratinolytic activity to proteases are, however, not well understood. Here, we investigated the structure-function relationship of a subtilisin-like S8 endopeptidase (FerB) from the thermophile Fervidobacterium pennivorans strain T. FerB was crystallized and its structure solved to 1.5 Å resolution, revealing an auto-processed state where the pro-peptide domain is non-covalently attached to the catalytic domain. The carboxyl group of the scissile peptide bond is coordinated in the active site within hydrogen bonding distance of the catalytic triad's serine residue. Unlike fervidolysin, no β-sandwich domains are present. However, a tyrosine-rich β-hairpin structure is found in the corresponding position within the FerB structure. Deletion of the β-hairpin reduced the protein's integrity and keratinase activity.