Cytochrome P450s are heme-containing monooxygenases involved in various physiological processes from archaea to mammals. They catalyze diverse types of reactions containing hydroxylations, epoxidations, dealkylations and heteroatom oxidations, which make P450s attractive industrial catalysts. P450s require electrons from NAD(P)H to convert molecular oxygen into reactive species. However, excess amounts of electron transfer partner proteins (more than 10 times) are necessary to obtain sufficient reaction rate and coupling efficiency and these properties are bottlenecks for practical applications of P450s.

Here, we show a site-specific branched fusion protein of P450 with its electron transfer proteins constructed using enzymatic cross-linking with a transglutaminase from Streptomyces mobaraensis (TGase). A branched fusion protein of P450 system, which was composed of one molecule each of P450 from Pseudomonas putida (P450cam) which catalyzes stereo-selective hydroxylation of d-camphor, putidaredoxin (Pdx) and putidaredoxin reductase (Pdr), showed higher catalytic activity (306 min-1) and coupling efficiency (99 %) than the reconstitution system due to the intramolecular electron transfer. Many substrates for P450s are highly hydrophobic and P450-catalyzed reactions are preferable to be conducted in nonaqueous media. The regeneration of NADH is also required due to its cost. We finally examined the hydroxylation of d-camphor by the fusion protein using NADH-regeneration driven by a hyper thermostable alcohol dehydrogenase in a reversed micella system. The hydroxylation of d-camphor and the regeneration of NADH were observed even in a reversed micellar system, while those were not observed using the reconstitution system.

This unique site-specific branched structure simply increased local concentration of proteins without serious loss of freedoms and activities of each protein. Therefore, enzymatic posttranslational protein manipulation can be a powerful alternative to conventional strategies for the creation of multi-component enzyme systems with novel proteinaceous architecture.