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Retuning Rieske-type oxygenases to expand substrate range.

Mahmood Mohammadi1, Jean-François Viger, Pravindra Kumar

  • 1Institut National de la Recherche Scientifique-Institut Armand-Frappier, Laval, Quebec H7V 1B7, Canada.

The Journal of Biological Chemistry
|June 10, 2011
PubMed
Summary
This summary is machine-generated.

Rieske-type oxygenases can be engineered to better degrade pollutants like dibenzofuran. Specific mutations enhance catalytic activity by altering the enzyme's active site and substrate-induced fit.

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Area of Science:

  • Biocatalysis
  • Enzyme Engineering
  • Environmental Biotechnology

Background:

  • Rieske-type oxygenases are valuable biocatalysts for pollutant degradation and chemical synthesis.
  • Expanding the substrate range of these enzymes is crucial for their practical applications.
  • Understanding enzyme evolution pathways informs protein engineering strategies.

Purpose of the Study:

  • To investigate how mutations in Rieske-type oxygenases affect their substrate range and catalytic efficiency.
  • To explore the structural and mechanistic basis for enhanced activity towards dibenzofuran.

Main Methods:

  • Site-directed mutagenesis was used to create BphAE variants (BphAE(p4) and BphAE(RR41)).
  • Enzyme kinetics were measured using steady-state kinetic analysis.
  • Crystal structures of enzyme variants were analyzed to understand structural changes.

Main Results:

  • BphAE(p4) and BphAE(RR41) showed 2x and 3x faster dibenzofuran metabolism than the wild-type BphAE(LB400).
  • Single T335A and double N338Q/L409F substitutions significantly enhanced catalytic activity.
  • Structural analysis revealed that T335A relieves catalytic cavity constraints, and N338Q/L409F alter substrate-induced conformational changes.

Conclusions:

  • Mutations altering catalytic cavity constraints or plasticity can expand enzyme substrate range.
  • Responsive induced fit mechanisms, influenced by mutations, are key to achieving efficient substrate transformation.
  • Enzyme engineering of Rieske-type oxygenases offers a pathway to improved biocatalysts for environmental and chemical applications.