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

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 th...

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Bibliographic Details
Published in:Protein engineering, design and selection design and selection, 2007-09, Vol.20 (9), p.453-459
Main Authors: Hirakawa, Hidehiko, Kamiya, Noriho, Tanaka, Tsutomu, Nagamune, Teruyuki
Format: Article
Language:English
Subjects:
branched structure
Camphor 5-Monooxygenase - chemistry
Camphor 5-Monooxygenase - metabolism
Catalysis
Cross-Linking Reagents - pharmacology
CYP101
cytochrome P450
Dose-Response Relationship, Drug
Electrons
Ferredoxins - chemistry
Homeodomain Proteins - metabolism
Mass Spectrometry
Models, Biological
Models, Molecular
Protein Binding
Pseudomonas putida
Pseudomonas putida - metabolism
site-specific cross-linking
Spectrophotometry, Ultraviolet
Trans-Activators - metabolism
transglutaminase
Transglutaminases - chemistry
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Summary: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.
ISSN:1741-0126
1741-0134
DOI:10.1093/protein/gzm045