Molecular Assembly of P450 with Ferredoxin and Ferredoxin Reductase by Fusion to PCNA

Proliferating cell nuclear antigen (PCNA) is a trimeric ringshaped protein that binds to dsDNA and acts as a scaffold for DNA-related enzymes, such as DNA polymerase and helicase. Although most PCNAs form homotrimers, three PCNAs found in Sulfolobus solfataricus form a heterotrimer. Fusion proteins between these PCNAs and functional proteins can act as nanoscale parts that can self-assemble to form a functional nanohybrid complex. Bacterial cytochrome P450 (P450), which is a promising biocatalyst, generally needs to accept electrons from NAD(P)H for its monooxygenase activity. The P450 interacts with a ferredoxin that is reduced by a specific ferredoxin reductase with NAD(P)H. Fusing these proteins with PCNAs can generate functional, stand-alone complexes of P450 and electron transfer-related proteins. Here we show that three PCNA proteins, fused with either a bacterial P450 or one of two electron transfer-related proteins, form a stable heterotrimeric complex (PCNA-utilized protein complex of P450 and its two electron transfer-related proteins, PUPPET). In PUPPET, P450 and the electron transfer-related proteins were in extremely close proximity to each other, enabling efficient electron transfer within the complex. As a result, PUPPET showed much higher catalytic activities compared with an equimolar mixture of the constituent components. The formation of complexes using PCNA could be a valuable strategy for the construction of complicated multienzymatic reactions, as well as for applications using electron transfer-related proteins. In vivo, proteins have sophisticated functions by 1) the formation of self-assembled complexes such as the proteasome, fatty-acid synthases and photosystems, and 2) intermolecular interactions with the assistance of biological molecules such as scaffold, anchoring and adaptor proteins that coordinate protein-kinase-related cell signaling. Microsomal P450s, which are involved in important physiological roles, such as xenobiotic detoxification, procarcinogen activation and steroid hormone synthesis, are anchored on the cytoplasmic surface of the endoplasmic reticulum membrane. This membrane anchoring facilitates interactions and electron transfers between P450s and membrane binding cytochrome P450 reductase, which is essential for P450-catalyzed reactions. In contrast, bacterial P450s, a lot of whose genes have been discovered during genome projects, are cytosolic proteins and generally require intermolecular electron transfer by two kinds of protein, ferredoxins and FAD-containing ferredoxin reductases, without any assistance from other biological molecules. Thus, higher concentrations of ferredoxins than dissociation constants are usually necessary for efficient intermolecular electron transfers to bacterial P450s, while the low concentration of the electron transfer proteins causes poor electron donation to the P450s and decreases their monooxygenase activities. Meanwhile, an artificial fusion P450, which was fused to specific electron transfer-related proteins by site-specific enzymatic crosslinking, showed a high catalytic activity due to intramolecular electron transfer. Artificial genetic fusion proteins of P450 and electron transfer protein(s) have also been developed to promote electron transfer. PCNA is a DNA sliding clamp that acts as a scaffold and interacts with many kinds of proteins involved in various cellular processes. Recently, heterotrimeric PCNAs have been found in crenarchaeota. 17, 18] The crenarchaeon S. solfataricus has three distinct PCNAs (PCNA1, PCNA2, and PCNA3). PCNA1 and PCNA2 first form a stable heterodimer, which then binds to PCNA3 to form a ring-shaped heterotrimer. Structural analyses have revealed that all of the PCNA C termini in the heterotrimer are exposed on the same side of the ring. These physiological and structural properties inspired us to fuse a bacterial P450 and its two electron transfer-related proteins, ferredoxin and ferredoxin reductase, to the C termini of the three PCNAs, thus engineering a heterotrimeric complex of P450 tightly juxtaposed to ferredoxin and enabling high local concentrations of ferredoxin for P450 and ferredoxin reductase. We constructed fusion proteins by genetically linking the C termini of PCNAs with the N termini of the proteins composing the P450 system of Pseudomonas putida, cytochrome P450 (P450cam, EC 1.14.15.1), putidaredoxin (PdX) and putidaredoxin reductases (PdR, EC 1.18.1), which are well-studied P450 and electron transfer proteins, respectively. 20] Three fusion proteins, PCNA1–PdR, PCNA2–PdX, and PCNA3–P450cam (Figure 1 A), were separately expressed and purified. Each fusion protein showed a characteristic cofactor-derived UV/Vis spectrum, which was similar to that of the respective component protein, PdR, PdX or P450cam (Figure S1 in the Supporting Information). These results suggested that the environments in the fusion proteins around cofactors, FAD, [2Fe–2S] cluster and heme, were similar to those in the component proteins and that each protein would retain activity after fusion to PCNAs. The function of each fusion protein was also examined separately. The PdX reducing activity of PCNA1–PdR (69.2 1.2 mm min ), which was directly measured by absorption change of PdX, was similar to that of wild-type PdR (73.8 [a] Dr. H. Hirakawa, Prof. T. Nagamune Department of Bioengineering, School of Engineering The University of Tokyo 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan) Fax: (+ 81) 3-5841-8657 E-mail : nagamune@bioeng.t.u-tokyo.ac.jp [b] Prof. T. Nagamune Department of Chemistry and Biotechnology, School of Engineering The University of Tokyo 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201000226.