Proliferating cell nuclear antigen (PCNA) is a trimeric ringshaped protein that binds to dsDNA and acts as a scaffold for DNA-related enzymes, [1] such as DNA polymerase and helicase. Although most PCNAs form homotrimers, three PCNAs found in Sulfolobus solfataricus form a heterotrimer. [2][3][4] 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, [5][6][7][8] 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, [9]

The catalytic activity of Staphylococcus aureus sortase A (SaSrtA) is dependent on Ca(2+), because binding of Ca(2+) to Glu residues distal to the active site stabilizes the substrate binding site. To obtain Ca(2+)-independent SaSrtA, we substituted two Glu residues in the Ca(2+)-binding pocket (Glu(105) and Glu(108)). Although single mutations decreased SaSrtA activity, mutations of both Glu(105) and Glu(108) resulted in Ca(2+)-independent activity. Kinetic analysis suggested that the double mutations affect the substrate binding site, without affecting substrate specificity. This approach will allow us to develop SaSrtA variants suitable for various applications, including in vivo site-specific protein modification and labeling.

A Staphylococcus aureus transpeptidase, sortase A (SrtA), catalyzes selective peptide/protein ligations that have been applied to cell imaging and protein engineering, while the ligations do not proceed to completion due to their reversibility. We successfully enhanced SrtA-mediated protein ligation through the formation of a β-hairpin around the ligation site.

A Staphylococcus aureus transpeptidase, sortase A (SrtA), which catalyzes a peptide ligation with high substrate specificity, is a useful tool to site-specifically attach proteinaceous/peptidic functional molecules to target proteins. However, its strong Ca(2+) dependency makes SrtA difficult for use under low Ca(2+) concentrations and in the presence of Ca(2+)-binding substances. To overcome this problem, we designed a SrtA mutant that Ca(2+)-independently demonstrates a high catalytic activity. The heptamutant (P94R/E105K/E108A/D160N/D165A/K190E/K196T), which resulted from a combination of known mutations at the Ca(2+) -binding site and around the substrate-binding site, successfully catalyzed a selective protein-protein ligation in the cytoplasm of Escherichia coli. Selective protein modification in living cells is a promising approach for investigating cellular events and regulating cell functions. This SrtA mutant may prove to be a versatile tool for adding new functionalities to proteins of interest by incorporating functional proteins and chemically modified peptides in living cells, which usually retain low Ca(2+) concentrations.