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Keywords:

  • biocatalysis;
  • cytochromes;
  • enzyme catalysis;
  • heme proteins;
  • high throughput

Abstract

Cytochromes P450 (P450s) are a family of haem-containing oxidases with considerable potential as tools for industrial biocatalysis. Organismal genomes are revealing thousands of gene sequences that encode P450s of as yet unknown function, the exploitation of which will require high-throughput tools for their isolation and characterisation. In this report, a ligationindependent cloning vector “LICRED” is described that enables the high-throughput generation of libraries of redox-self-sufficient P450s by fusing a range of P450 haem domains to the reductase of P450RhF (RhF-Red) in a robust and generically applicable way. Cloning and expression of fusions of RhF-Red with the haem domains of P450cam and P450-XplA resulted in soluble, active, redox-self-sufficient, chimeric enzymes. In vitro studies also revealed that electron transfer from NADPH to haem was primarily intramolecular. The general applicability of the LICRED platform was then demonstrated through the creation of a library of RhF-Red fusion constructs by using the diverse complement of P450 haem domains identified in the genome of Nocardia farcinica. The resultant fusion-protein library was then screened against a panel of substrates; this revealed chimeric enzymes competent for the hydroxylation of testosterone and methyltestosterone, and the dealkylation of 7-ethoxycoumarin.