9QBD

Crystal structure of Brachyspira hampsonii PadR with Leu88 replaced by 3-aminotyrosine

Summary for 9QBD

• Entry DOI: 10.2210/pdb9qbd/pdb
• Descriptor: PadR-family transcriptional regulator (2 entities in total)
• Functional Keywords: transcriptional regulator, padr family, unnatural amino acid, 3-aminotyrosine, dna binding protein
• Biological source: Brachyspira hampsonii
• Total number of polymer chains: 2
• Total formula weight: 28884.25

Authors

Thunnissen, A.M.W.H.,Brouwer, B.,Roelfes, G. (deposition date: 2025-03-02, release date: 2026-03-11, Last modification date: 2026-05-13)

Primary citation

Brouwer, B.,Thunnissen, A.W.H.,Rozeboom, H.J.,Roelfes, G.
Exploring PadR Proteins for Artificial Enzyme Design.
Chembiochem, 27:e70308-e70308, 2026

PubMed Abstract: 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.

PubMed: 42047317
DOI: 10.1002/cbic.70308

Experimental method

X-RAY DIFFRACTION (2.1 Å)