Enzyme Engineering

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Hidehiko Hirakawa
Assistant Professor
Departmenet of Chemistry and Biotechnology

Academic Degrees
B.S., 2000, The University of Tokyo
Ph.D., 2005, The University of Tokyo
Postdoctoral Fellow, 2005-2007, University of Kansas
Project Postdoctoral fellow, 2007-2009, The University of Tokyo
Project Assistant Professor, 2009-2010, The University of Tokyo

Enzymes are and energy-saving and environmentally-friendly catalysts because they show high selectivities (reaction selectivity, regioselectivity and stereoselectivity) in aqueous solution under physiological conditions. We are trying to develop novel enzymes (system) that catalyze complexed reactions by approaching away from active sites of enzymes.

PCNA-utilized protein complex formation
PCNAs, which play a role as scaffold for DNA replication/modification enzymes, are ring-shaped trimeric proteins. We are trying to enhance natural function of protein and to express novel function of protein by selective complex formation using selef-assembly of PCNA. We have already shown that compelx formation of cytochrome P450 and its electron transfer proteins using PCNA improved cytochrome P450's activity. This research was supported by MEXT KAKENHI 21760633.

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Hirakawa and Nagamune, ChemBioChem, 11, 1517-1520 (2010).

Cytochrome P450 (pink) requires two electrons for its catalytic activity. Electrons are transfered through ferredoxin (oxidized, blue; reduced, cyan) and ferredoxin reductase (green). Efficient electron transfer requires excess amounts of ferredoxin and fereedoxin reductase.

Heterotrimerization of P450, ferredoxin and ferredoxin reductase using PCNA increases the local concentration of ferredoxin. The heterotrimer show high activity by itself due to the efficient electron transfer within the complex.

Transglutaminase-mediated construction of branched fusion protein
Transglutaminase (TGase) catalyzes cross-linking between side chains of glutamine and lysine residues. We successfully constructed a branched fusion protein using a site-specific cross-linking by TGase. A branched fusion P450cam, which is consisted of P450cam and its electron transfer proteins, showed a high catalytic activity due to intramolecular electron transfer.

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Hirakawa et al, Protein Eng. Des. & Sel., 20, 453-459 (2007).

Enhancement of peptide ligation using beta-hairpin structure
In enzymatic reactions, it is essential that substrates conformationally fit into the substrate binding sites of the enzymes to control the spatial arrangements and orientations of the substrates in the active site. These conformational restrictions determine the substrate specificities of enzymes. We hypothesize that the introduction of a rigid secondary structure to ligation products will inhibit products' binding to SrtA and that a shift in the equilibrium to formation of a SrtA-unrecognized product will increase the ligation yield. We successfully enhanced SrtA-mediated protein ligation through the formation of a b-hairpin around the ligation site.

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Yamamura et al, Chem. Commun., 47, 4742-4744 (2011).

Publications
Y. Yamamura, H. Hirakawa*, S. Yamaguchi and T. Nagamune*, (2011) "Enhancement of sortase A-mediated protein ligation by inducing a beta-hairpin structure around the ligation site", Chem. Commun., 47, 4742-4744 (2011).
DOI: 10.1039/C0CC05334A, PMID: 21409251

T. Reed, G.H. Lushington, Y. Xia, H. Hirakawa, D.M. Travis, M. Mure*, E.E. Scott* and J. Limburg, (2010) "Crystal structure of histamine dehydrogenase from Nocardioides simplex", J. Biol. Chem. 285, 25782-25791.
DOI: 10.1074/jbc.M109.084301, PMID: 20538584

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

H. Hirakawa, N. Kamiya, Y. Kawarabayasi and T. Nagamune*, (2010) "Artificial Self-sufficient P450 in Reversed Micelles", Molecules, 15, 2935-2948. Open access
DOI: 10.3390/molecules15052935 PMID: 20657456

T.M. Reed, H. Hirakawa, M. Mure, E.E. Scott* and J. Limburg*, (2008) "Expression, purification, crystallization and preliminary X-ray studies of histamine dehydrogenase from Nocardiodes simplex", Acta Cryst. F64, 785-787. Open access
DOI: 10.1107/S1744309108023336, PMID: 18765904

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. Open access
DOI: 10.1093/protein/gzm045, PMID: 17827502

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

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

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