Cytochrome P450s are heme-containing monooxygenases that require electron transfer proteins for their catalytic activities. They prefer hydrophobic compounds as substrates and it is, therefore, desirable to perform their reactions in non-aqueous media. Reversed micelles can stably encapsulate proteins in nano-scaled water pools in organic solvents. However, in the reversed micellar system, when multiple proteins are involved in a reaction they can be separated into different micelles and it is then difficult to transfer electrons between proteins. We show here that an artificial self-sufficient cytochrome P450, which is an enzymatically crosslinked fusion protein composed of P450 and electron transfer proteins, showed micelle-size dependent catalytic activity in a reversed micellar system. Furthermore, the presence of thermostable alcohol dehydrogenase promoted the P450-catalyzed reaction due to cofactor regeneration.

H. Hirakawa, N. Kamiya, Y. Kawarabayasi and T. Nagamune*, (2010) "Artificial Self-sufficient P450 in Reversed Micelles", Molecules, 15, 2935-2948.

DOI: 10.3390/molecules15052935, PMID: 20657456

A NAD⁺-dependent medium-chain alcohol dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernix K1 was expressed in Escherichia coli and purified. The recombinant enzyme was a homotetramer of molecular mass 1.6 x 10² kDa. The optimum pH for the oxidative reaction was around 10.5 and that for the reductive reaction was around 8.0. The enzyme had a broad substrate specificity including aliphatic and aromatic alcohols, aliphatic and aromatic ketones, and benzylaldehyde. This enzyme produced (S)-alcohols from the corresponding ketones. The enzyme was thermophilic and the catalytic activity increased up to 95 degrees C. It maintained 24% of the original catalytic activity after incubation for 30 min at 98 degrees C, indicating that this enzyme is highly thermostable.

H. Hirakawa, N. Kamiya, Y. Kawarabayashi and T. Nagamune*, (2004) "Properties of an Alcohol dehydrogenase from the Hyperthermophilic Archaeon Aeropyrum pernix K1", J. Biosci. Bioeng. 97 (3), 202-206.

DOI: 10.1016/S1389-1723(04)70191-7, PMID: 16233615

The regioselective reduction of androstandione to androsterone by 3α-hydroxysteroid dehydrogenase (HSDH) from Pseudomonas testosteroni was coupled to a cofactor (NADH) regeneration system with the oxidation of ethanol by yeast alcohol dehydrogenase (YADH) in sodium dioctyl sulfosuccinate (AOT)/isooctane reversed micelles. Each reaction was dominated by the system’s water content (W₀=[H2O]/[AOT]). The catalytic activity of YADH increased monotonically with increasing W₀, whereas HSDH showed bell-shaped dependency on W₀. Using a reversed micellar system increased stability of both enzymes in comparison with an aqueous system and prolonged NADH-regeneration; namely, a seven-fold increase in the total turnover number of NADH was achieved. These results suggest that the reversed micellar system is a promising choice for conjugate enzymatic reactions with dehydrogenases in a nonaqueous media.

H. Hirakawa, N. Kamiya, T. Yata and T. Nagamune*, (2003) "Regioselective reduction of a steroid in a reversed micellar system with enzymatic NADH-regeneration", Biochem. Eng. J. 16, 35-40.

DOI: 10.1016/S1369-703X(03)00019-6

An alcohol dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernix was activated by water-miscible organic solvents. This activation was influenced by the kind and the concentration of the added organic solvents. The k_cat was increased by a factor of over ten when the mole fraction of acetonitrile was 0.1. This effect was large when organic solvents with large log P values were added. In fact, the k_cat showed a strong positive correlation with the log P value of the mixed solvent at a constant mole fraction of water, while it was not affected by the kind of organic solvents added. Both the activation enthalpy and the entropy decreased with an increase in log P. The contribution of the activation enthalpy to the free energy of activation was larger than that of the activation entropy, and the free energy of activation decreased with an increase in log P.

H. Hirakawa, N. Kamiya, Y. Kawarabayashi and T. Nagamune*, (2005) "Log P effect of organic solvents on a thermophilic alcohol dehydrogenase", Biochemica Biophysica Acta 1748, 94-98.

DOI: 10.1016/j.bbapap.2004.12.007, PMID: 15752697

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.

H. Hirakawa, N. Kamiya, T. Tanaka and T. Nagamune*, (2007) "Intramolecular electron transfer in a cytochrome P450cam system with a site-specific branched structure", Protein Eng. Des. Sel., 20, 453-459.

DOI: 10.1093/protein/gzm045, PMID: 17827502

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 bottom-up functional nanoarchitectures. Bacterial cytochrome P450 (P450), which is a promising biocatalyst, needs to accept electrons for its monooxygenase activity, interacting with a ferredoxin that is reduced by a specific ferredoxin reductase. Fusing these proteins with PCNAs can generate functional stand-alone complexes of P450 and electron transfer proteins, although each component protein is, itself, unfunctional. Here we show that three PCNA proteins, fused with either a bacterial P450 or one of two electron transfer proteins, formed a stable heterotrimeric complex (PCNA-utilized potent heterotrimeric complex of P450 and its electron transfer proteins, PUPPET). In PUPPET, P450 and the electron transfer 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 utilizing PCNA could be a valuable strategy for the construction of complicated multienzymatic reactions, as well as for applications using electron transfer related proteins.

H. Hirakawa and T. Nagamune*, (2010) "Molecular Assembly of P450 with Ferredoxin and Ferredoxin Reductase by Fusion to PCNA", ChemBioChem, 11, 1517-1520.

DOI: 10.1002/cbic.201000226, PMID: 20607777

Plasmids constructed in this study are available from Addgene (www.addgene.org/browse/article/10354/).

The catalytic activity of Staphylococcus aureus sortase A (SaSrtA) is dependent on Ca²⁺, because binding of Ca²⁺ to Glu residues distal to the active site stabilizes the substrate binding site. To obtain Ca²⁺-independent SaSrtA, we substituted two Glu residues in the Ca²⁺-binding pocket (Glu¹⁰⁵ and Glu¹⁰⁸). Although single mutations decreased SaSrtA activity, mutations of both Glu¹⁰⁵ and Glu¹⁰⁸ resulted in Ca²⁺-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.

H. Hirakawa*, S. Ishikawa and T. Nagamune, (2012) "Design of Ca²⁺-independent Staphylococcus aureus sortase A mutants", Biotechnol. Bioeng., 109, 2955-2961.

DOI: 10.1002/bit.24585 , PMID: 22729808

Plasmids constructed in this study are available from Addgene (www.addgene.org/browse/article/10128/).

Most of the bacterial cytochrome P450 s require two kinds of electron transfer proteins, ferredoxin and ferredoxin reductase, and thus P450 s do not show catalytic activity by themselves. A microbial transglutaminase-mediated site-specific cross-linking enables the formation of fusion P450 protein with a branched structure, which is generated from a genetic fusion protein of P450–ferredoxin reductase and ferredoxin, an interactive nanoscale protein structure. This fusion P450 system is self-sufficient due to intramolecular electron transfer, which means the system does not require additional electron-transferring proteins. Because some components of bacterial cytochrome P450 system are interchangeable, this self-sufficient system can be applied to non-natural combination of P450 and electron transfer proteins from different species of bacteria.

H. Hirakawa and T. Nagamune*, (2011) "Nanoscale-Engineered Cytochrome P450 System with a Branch Structure", NANOSCALE BIOCATALYSIS, Methods in Molecular Biology, 2011, Volume 743, 1-16, DOI: 10.1007/978-1-61779-132-1_1

In nature, enzymes often form multienzyme complexes to enhance their catalytic efficiencies and, ­moreover, evolve into genetically fused multidomain enzymes. Inspired by a natural fusion cytochrome P450 (P450) containing a monooxygenase domain and a reductase domain, we have developed a heterotrimeric protein-utilized method to form a multienzyme complex composed of a bacterial P450 and its catalytically essential two redox proteins. Three distinct proliferating cell nuclear antigens (PCNAs) from Sulfolobus solfataricus, each of which can be separately expressed, spontaneously form a heterotrimer. Fusion to the PCNAs enables complex formation of a bacterial P450 and two redox proteins through the self-assembling of the PCNAs and enhances the activity due to efficient electron transfer in the complex. This PCNA-mediated multienzyme complex formation will be available for other multienzyme reactions.

H. Hirakawa and T. Nagamune*, (2013) "Nanoscale-Engineered Cytochrome P450 System with a Branch Structure", Enzyme Engineering, Methods in Molecular Biology Volume 978, 2013, pp 149-163.

DOI: 10.1007/978-1-62703-293-3_11

In nature, many enzymes participating in multienzyme reactions are often assembled to enhance efficiencies of multiple reactions. Therefore, much attention has been focused on self-assembly of multiple enzymes fused with a protein/peptide that interacts with a specific protein to enhance artificial multienzyme reactions. Sulfolobus solfataricus proliferating cell nuclear antigen (PCNA) is a ring-shaped symmetric heterotrimer consisting of PCNA1, PCNA2 and PCNA3. Multiple enzymes can be co-localized on the PCNA ring by fusing them to the C-termini of the three PCNA subunits. However, an advantage of the specific non-covalent complex formation is inextricably associated with the disadvantage of its concentration-dependent dissociation. In this study, disulfide bonds were introduced between the PCNA subunits by Cys substitution at the sites neighboring the interface for heterotrimerization. Selective intersubunit disulfide bond formation between PCNA1 and PCNA3 and between PCNA2 and PCNA3 by a natural oxidizing reagent successfully stabilized an artificial multienzyme complex, which is composed of a bacterial cytochrome P450 and its two redox partner proteins. The covalent stabilization of the multienzyme complex enhanced its cytochrome P450 activity because of the absence of inactive dissociated components.

H. Hirakawa*, A. Kakitani and T. Nagamune*, (2013) "Introduction of Selective Intersubunit Disulfide Bonds into Self-assembly Protein Scaffold to Enhance an Artificial Multienzyme Complex's Activity", Biotechnol. Bioeng., 110: 1858–1864. doi: 10.1002/bit.24861.

Plasmids constructed in this study are available from Addgene (www.addgene.org/browse/article/10359/).