Intramolecular electron transfer in a cytochrome P450cam system with a site-specific branched structure

Protein Eng Des Sel. 2007 Sep;20(9):453-9. doi: 10.1093/protein/gzm045. Epub 2007 Sep 7.

Abstract

Cytochrome P450 (P450) is an attractive oxygenase due to the diverse catalytic reactions and the broad substrate specificity. Class I P450s require an excess concentration (more than 10 times) of iron-sulfur proteins, which transfer electrons to P450s, to attain the maximum catalytic activity and this requirement is a critical bottleneck for practical applications. Here, we show a site-specific branched fusion protein of P450 with its electron transfer proteins using enzymatic cross-linking with transglutaminase. A branched fusion protein of P450 from Pseudomonas putida (P450cam), which was composed of one molecule each of P450cam, putidaredoxin (Pdx) and Pdx reductase, showed higher catalytic activity (306 min(-1)) and coupling efficiency (99%) than the equimolar reconstitution system due to the intramolecular electron transfer. The unique site-specific branched structure simply increased local concentration of proteins without denaturation of each protein. Therefore, enzymatic post-translational protein manipulation can be a powerful alternative to conventional strategies for the creation of multicomponent enzyme systems with novel proteinaceous architecture.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Camphor 5-Monooxygenase / chemistry*
  • Camphor 5-Monooxygenase / metabolism
  • Catalysis
  • Cross-Linking Reagents / pharmacology
  • Dose-Response Relationship, Drug
  • Electrons
  • Ferredoxins / chemistry
  • Homeodomain Proteins / metabolism
  • Mass Spectrometry
  • Models, Biological
  • Models, Molecular
  • Protein Binding
  • Pseudomonas putida / metabolism
  • Spectrophotometry, Ultraviolet
  • Trans-Activators / metabolism
  • Transglutaminases / chemistry

Substances

  • Cross-Linking Reagents
  • Ferredoxins
  • Homeodomain Proteins
  • Trans-Activators
  • pancreatic and duodenal homeobox 1 protein
  • putidaredoxin
  • Camphor 5-Monooxygenase
  • Transglutaminases