Exploring PadR Proteins for Artificial Enzyme Design

The development of artificial enzymes through incorporation of new-to-nature catalytic functionality into protein scaffolds has emerged as a powerful approach to expand the biocatalytic repertoire. Inspired by the success of Lactococcal multidrug resistance regulator (LmrR), a transcriptional regulator protein, whose unique scaffold has been used for the design of a range of artificial enzymes, we performed a bioinformatics study in an effort to expand the scope of protein scaffolds for artificial enzyme design with other LmrR-like proteins. LmrR belongs to the phenolic acid decarboxylase transcriptional regulator (PadR) subfamily 2 (PadR-s2) and exhibits an unusual open pore with promiscuous binding capabilities. Using genome mining and homology modeling, we identified six previously uncharacterized PadR-s2 proteins and experimentally evaluated them as protein scaffolds for the design of artificial Friedel-Crafts (FC) alkylases. Two of the candidates, Lactococcus fujiensis (LCf) PadR and Brachyspirahampsonii (Bh) PadR, could be applied in the iminium-promoted FC-alkylation using genetically incorporated noncanonical amino acids p-aminophenylalanine or 3-aminotyrosine as catalytic residues. Interestingly, contrary to homology models, AlphaFold predictions of the PadR-s2 candidates and X-ray crystallography of BhPadR and a variant incorporating 3-aminotyrosine revealed closed-pore structures. Our findings thus demonstrate that an open-pore structure like LmrR is not a prerequisite for designing artificial FC-alkylases and introduce two new PadR-s2 scaffolds for future application.