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A hydroquinone-specific screening system for directed P450 evolution.

Alexandra M Weingartner1, Daniel F Sauer1, Gaurao V Dhoke1

  • 1Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany.

Applied Microbiology and Biotechnology
|September 8, 2018
PubMed
Summary
This summary is machine-generated.

Researchers developed a new screening system to identify improved cytochrome P450 (CYP) enzyme variants for producing hydroquinones. A novel P450 BM3 variant, AW2, showed a 70-fold increase in trimethylhydroquinone formation, advancing biocatalysis for aromatic compound oxidation.

Keywords:
Aromatic hydroxylationDirected evolutionHydroquinoneP450 BM3Protein engineeringScreening assay

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Area of Science:

  • Biocatalysis and Enzyme Engineering
  • Organic Chemistry
  • Molecular Biology

Background:

  • Direct benzene hydroxylation to hydroquinone (HQ) is challenging for chemical catalysts.
  • Cytochrome P450 (CYP) monooxygenases oxidize aromatic compounds using dioxygen.
  • Protein engineering enhances CYP activity, specificity, and stability, but requires effective screening.

Purpose of the Study:

  • To develop a novel screening system for identifying P450 variants that produce hydroquinones.
  • To quantify hydroquinone production using a 4-nitrophenylacetonitrile (NpCN) assay.
  • To discover improved P450 BM3 variants for trimethylhydroquinone (TMHQ) formation.

Main Methods:

  • Development of a hydroquinone quantification assay based on NpCN interaction under alkaline conditions.
  • Implementation of the NpCN assay in a 96-well plate format for high-throughput screening.
  • Screening of a P450 BM3 saturation mutagenesis library at amino acid position A330.

Main Results:

  • The NpCN assay demonstrated a low detection limit (5 μM) and a broad linear range (5–250 μM) for hydroquinone quantification.
  • The assay successfully quantified dihydroxylated aromatic compounds, including hydroquinones, catechols, and benzoquinones.
  • A new P450 BM3 variant, AW2 (R47Q, Y51F, I401M, A330P), was identified, exhibiting a 70-fold increase in TMHQ formation compared to wild type.

Conclusions:

  • A robust and sensitive NpCN-based screening system for hydroquinone-producing P450s was established.
  • The P450 BM3 variant AW2 represents a significant improvement in TMHQ production, surpassing previously reported variants.
  • This work facilitates the discovery of engineered enzymes for efficient biocatalytic hydroxylation of aromatic compounds.