Supramolecular protein assembly supports immobilization of a cytochrome P450 monooxygenase system as water-insoluble gel.
Diverse applications of the versatile bacterial cytochrome P450 enzymes (P450s) are hampered by their requirement for the auxiliary proteins, ferredoxin reductases and ferredoxins, that transfer electrons to P450s. Notably, this limits the use of P450s as immobilized enzymes for industrial purposes. Herein, we demonstrate the immobilization of a bacterial P450 and its redox protein partners by supramolecular complex formation using a self-assembled heterotrimeric protein. Employment of homodimeric phosphite dehydrogenase (PTDH) for cross-linking "proliferating cell nuclear antigen-utilized protein complex of P450 and its two electron transfer-related proteins" (PUPPET) yielded a gelling PUPPET-PTDH system capable of regenerating NADH for electron supply owing to its phosphite oxidation activity. The protein gel catalyzed monooxygenation in the presence of phosphite and NAD(+). The gel was completely water-insoluble and could be reused. This concept of oligomeric protein-insolubilized enzymes can be widely applied to various multienzymatic reactions such as cascade reactions and coupling reactions.
Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Cheau Yuaan
CY
Tan
Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Hidehiko
H
Hirakawa
1] Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan [2] Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Teruyuki
T
Nagamune
D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004579:Electron Transport; D004800:Enzymes, Immobilized; D005782:Gels / Q000737:chemistry; D007700:Kinetics; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D010084:Oxidation-Reduction; D010101:Oxygen Consumption; D055503:Protein Multimerization; D012995:Solubility
D004800:Enzymes, Immobilized; D005782:Gels; D046912:Multiprotein Complexes; D003577:Cytochrome P-450 Enzyme System; D009247:NADH, NADPH Oxidoreductases; C429539:NAD phosphite oxidoreductase