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Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Published on: July 25, 2013
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Exploring PadR Proteins for Artificial Enzyme Design
Bart Brouwer1, Andy-Mark W H Thunnissen2, Henriette J Rozeboom2
1Stratingh Institute for Chemistry, University of Groningen, Groningen, Netherlands.
Chembiochem : a European Journal of Chemical Biology
|April 28, 2026
Summary
Researchers explored new protein scaffolds for artificial enzyme design, inspired by the LmrR protein. They identified two novel PadR-s2 scaffolds, demonstrating their utility in creating artificial Friedel-Crafts alkylases, expanding biocatalysis possibilities.
Area of Science:
- Biochemistry
- Protein Engineering
- Synthetic Biology
Background:
- Artificial enzymes expand biocatalysis by incorporating novel functions into protein scaffolds.
- The transcriptional regulator Lactococcal multidrug resistance regulator (LmrR) is a successful scaffold for artificial enzyme design.
- LmrR belongs to the phenolic acid decarboxylase transcriptional regulator subfamily 2 (PadR-s2) and has an open-pore structure.
Purpose of the Study:
- To identify and evaluate new LmrR-like protein scaffolds for artificial enzyme design.
- To expand the repertoire of available scaffolds beyond LmrR.
- To investigate the suitability of PadR-s2 proteins as scaffolds for artificial Friedel-Crafts (FC) alkylases.
Main Methods:
- Bioinformatic genome mining to identify uncharacterized PadR-s2 proteins.
- Homology modeling and AlphaFold prediction for structural analysis.
- Experimental evaluation of identified proteins as scaffolds for artificial FC alkylases using genetically incorporated noncanonical amino acids.
- X-ray crystallography for detailed structural characterization.
Main Results:
- Six previously uncharacterized PadR-s2 proteins were identified.
- Two candidates, Lactococcus fujiensis (LCf) PadR and Brachyspirahampsonii (Bh) PadR, were successfully used as scaffolds for artificial FC alkylases.
- The catalytic activity involved iminium-promoted FC-alkylation using p-aminophenylalanine or 3-aminotyrosine.
- Structural analysis revealed closed-pore structures, contrasting with initial homology models.
Conclusions:
- Open-pore structure is not essential for designing artificial FC-alkylases.
- LCf PadR and Bh PadR are novel and effective scaffolds for artificial enzyme development.
- These findings introduce new protein scaffolds for expanding biocatalytic applications.
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