earticle

영어

Proliferating cell nuclear antigen (PCNA) is a trimeric ring‐shaped protein that binds to dsDNA and acts as a scaffold for DNA‐related enzymes, such as DNA polymerase and helicase. Although most of PCNAs form homotrimers, three PCNAs found in Sulfolobus solfataricus form a heterotrimer. Fusion proteins between these PCNAs and functional proteins can act as nano‐scale parts, which can self‐assemble to form functional nano‐hybrid complex.
Bacterial cytochrome P450 (P450), which is a promising biocatalyst, generally needs to accept electrons from NAD(P)H for its monooxygenase activity, interacting 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 P450 from Pseudomonas putida (P450cam) or one of two electron transfer related proteins (Pdx, PdR), formed a stable heterotrimeric complex (PCNA‐Utilized Protein Complex of P450 and Its Two Electron Transfer Related Proteins, PUPPET). In PUPPET, P450cam, PdX and PdR were in extremely close proximity to each other, enabling efficient electron transfer within the complex (Fig. 1). As a result, PUPPET showed about 60‐fold higher catalytic activities compared with an equimolar mixture of the constituent components. The formation of complexes utilizing PCNA could be a valuable strategy for the construction of complicated multi‐enzymatic reactions, as well as for applications using electron transfer related proteins.