title: Intramolecular electron transfer in a cytochrome P450cam system with a site-specific branched structure.
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.
authors.affiliation: Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
authors.forename: Hidehiko
authors.initials: H
authors.lastname: Hirakawa
authors.forename: Noriho
authors.initials: N
authors.lastname: Kamiya
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Tanaka
authors.forename: Teruyuki
authors.initials: T
authors.lastname: Nagamune
mesh_terms: D019475:Camphor 5-Monooxygenase / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004583:Electrons; D005288:Ferredoxins / Q000737:chemistry; D018398:Homeodomain Proteins / Q000378:metabolism; D013058:Mass Spectrometry; D008954:Models, Biological; D008958:Models, Molecular; D011485:Protein Binding; D016958:Pseudomonas putida / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D015534:Trans-Activators / Q000378:metabolism; D011503:Transglutaminases / Q000737:chemistry