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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.

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Summary
This summary is machine-generated.

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.

Keywords:
PadR‐s2artificial enzymebiocatalysisnoncanonical amino acidprotein scaffold

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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.