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

Hidehiko Hirakawa, Noriho Kamiya, Tsutomu Tanaka, Teruyuki Nagamune

Research output: Contribution to journalArticlepeer-review

44 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)453-459
Number of pages7
JournalProtein Engineering, Design and Selection
Issue number9
Publication statusPublished - Sept 2007

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Molecular Biology


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