PEDS Advance Access originally published online on September 7, 2007
Protein Engineering Design and Selection 2007 20(9):453-459; doi:10.1093/protein/gzm045
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Intramolecular electron transfer in a cytochrome P450cam system with a site-specific branched structure
1Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan 2Department of Applied Chemistry, Graduate School of Engineering and Center for Future Chemistry, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan
3 To whom correspondence should be addressed. E-mail: nagamune{at}bio.t.u-tokyo.ac.jp
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.
Keywords: branched structure/cytochrome P450/CYP101/site-specific cross-linking/transglutaminase
Received April 9, 2007; revised July 17, 2007; accepted July 17, 2007.
Abbreviations: His6-P450cam WT, His6-tagged P450cam at the N-terminus of the wild-type P450cam; His6-P450cam mutant, His6-tagged P450cam at the N-terminus of a mutant P450cam; C73S/C85S Pdx, the Cys73Ser/Cys85Ser mutant of Pdx; Qlinker, a peptide sequence including a reactive glutamine residue for TGase; CKtag, a peptide sequence including a reactive lysine residue for TGase, Pdr–Qlinker–P450cam, a fusion protein between Pdr and P450cam with Qlinker; Pdx–CKtag, Pdx fused with CKtag at the C-terminus of C73S/C85S Pdx; bRXC, branched Pdr–Pdx–P450cam triple fusion protein.