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 LmrR, a transcriptional regulator protein which 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 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 Brachyspira hampsonii (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.