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Cited by 1Enzyme Catalysis and ImmobilizationProtein Structure and DynamicsBiochemical and Structural Characterization

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Exploring PadR Proteins for Artificial Enzyme Design

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Brouwer, Bart, Thunnissen, Andy-Mark W. H., Rozeboom, Henriette J., Roelfes, Gerard·2026·ChemBioChem
DOI 10.1002/cbic.70308

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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.

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Cites

  • Highly accurate protein structure prediction with AlphaFold

    Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., Tunyasuvunakool, K., Bates, R., Žídek, A., Potapenko, A., et al. (2021) · Nature · doi:10.1038/s41586-021-03819-2 · Cited by 43,764 · cited 1×

    “AlphaFold [39, 40] was used to predict the structures of these putative PadR proteins starting from their amino acid sequences.”

  • Impact of protein conformational diversity on AlphaFold predictions

    Saldaño, T., Escobedo, N., Marchetti, J., Zea, D. J., Mac Donagh, J., Velez Rueda, A. J., Gonik, E., Melani, A. G., Novomisky Nechcoff, J., Salas, M. N., et al. (2021) · doi:10.1101/2021.10.27.466189 · Cited by 196 · cited 1×

    “Furthermore, potential conformational dynamics are not sam­ pled by these static structure predictions [43, 44], and similarly to LmrR, these PadR proteins might exhibit a degree of con­ formational flexibility [45–47].”

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Metadata

Authors
Brouwer, B., Thunnissen, A.-M. W. H., Rozeboom, H. J., & Roelfes, G.
Affiliations
  • University of Groningen (NL)
  • Groningen Biomolecular Sciences and Biotechnology Institute University of Groningen Groningen Netherlands
  • Stratingh Institute for Chemistry University of Groningen Groningen Netherlands
ORCID
Venue
ChemBioChem
Publisher
Wiley
Published
2026-04
Volume
27
Issue
8
Pages
e70308-e70308
ISSN
  • 1439-4227 (ISSN-L)
  • 1439-7633
DOI
10.1002/cbic.70308
OpenAlex
W7158208226
Cited by
1
References
2
Citations by year
2026: 1
FWCI
2.07
Type
journal article
Language
en
Open access
Licence
Locations
  • ChemBioChem (publishedVersion, cc-by)
  • ChemRxiv (acceptedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • University of Groningen research database (University of Groningen / Centre for Information Technology) (publishedVersion, cc-by)
Funders
  • NWO (OCENW.KLEIN.143)
  • ERC (885396)
  • ESRF (MX-2649)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Structural Characterization
Keywords
enzyme mimics, LmrR, protein scaffolds, transcription factors, p-aminophenylalanine, unnatural amino acids, open pore structure, closed-pore structure, genome mining, homology modeling, AlphaFold predictions
MeSH terms
Bacterial Proteins, Carboxy-Lyases, Models, Molecular, Protein Engineering

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CoChat. (2026). Citation check: Exploring PadR Proteins for Artificial Enzyme Design. https://app.cochat.ai/papers/10.1002/cbic.70308
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Brouwer, B., Thunnissen, A.-M. W. H., Rozeboom, H. J., & Roelfes, G. (2026). Exploring PadR Proteins for Artificial Enzyme Design. ChemBioChem. https://doi.org/10.1002/cbic.70308