(66c) PCNA-Mediated Gelation and Immobilization of Multi-Enzyme Complex

Authors: 
Nagamune, T. - Presenter, The University of Tokyo
Hirakawa, H., The University of Tokyo



In bacterial P450 systems, FdXs act as shuttle molecules between FdRs and P450s. Therefore, co-immobilization of FdRs, FdXs and P450s using carries or cross-linking reagents inhibits electron transfer to P450s from NADH through FdRs and FdXs. Here, we overcome the problem by supermolecular assembly of the Proliferating cell nuclear antigen (PCNA), which is a trimeric ring-shaped protein that encircles dsDNA. FdRs, FdXs and P450s were fused at the C-terminus of each PCNA subunit [1] and the three fusion proteins were homodimerized by genetically linking to a homodimeric phosphite dehydrogenase (PTDH) at the N-terminus. An equimolar mixture of the three fusion proteins, PTDH-PCNA1-FdR, PTDH-PCNA2-FdX and PTDH-PCNA3-P450 immediately gelated because the three fusion proteins were connected by the PCNA heterotrimerization. The gel was stabilized by introduction of selective disulfide bonds between PCNA subunits, which was achieved using the Cys mutants of the PCNA subunits [2]. The gel of FdX-FdR-P450 protein complex showed the monooxygenase activity and was available for a repeated batch reaction. Furthermore, because PTDH can regenerate NADH with phosphite, the gel catalyzed the monooxygenation reaction consuming phosphite in the presence of NAD+. This PCNA-mediated gelation, that is, insolubilization is a promising immobilization method for single enzymes as well as multienzyme complexes because each enzyme can completely retain its activity.

References

[1] H. Hirakawa and T. Nagamune, (2010) ChemBiochem 11: 1517-1520.

[2] H. Hirakawa, A. Kakitani and T. Nagamune (2013) Biotechnol. Bioeng. in press.