Artificial Self‐Sufficient Cytochrome P450 Containing Multiple Auxiliary Proteins Demonstrates Improved Monooxygenase Activity

Abstract

Most bacterial cytochrome P450 monooxygenases (P450s) do not work alone because their active species is generated by two electrons supplied through two separate auxiliary proteins. Artificial "self-sufficient" P450s, in which one molecule each of the two auxiliary proteins is arranged close to the P450s, have been developed but have not achieved the maximum catalytic turnover numbers of the P450s. In this study, the Pseudomonas putida P450 (P450cam) is assembled with multiple molecules of its auxiliary proteins, putidaredoxin (PdX) and putidaredoxin reductase (PdR), by fusion to a heterotrimeric protein. In the assembled P450cam containing one PdX and one PdR, kinetic analysis reveales that the catalytic cycle of P450cam is suspended twice awaiting the reduction of PdX by PdR. An increase in the number of PdR molecules stimulated the PdX reduction process. Assembly with two PdXs allows one PdX to be reduced during the binding of the other PdX to P450cam for the first electron transfer, eliminating one waiting step. Finally, P450cam assembled with two PdXs and three PdRs showes 92% of the maximum activity of free P450cam. Therefore, assembly with multiple molecules of auxiliary proteins will facilitate in vitro biotechnological applications of the P450s.

Biomimetic and Bioinspired Biotechnology

“…Tanaka et al describe a lipid tubule fabrication technique using MamY protein responsible for megnetosome vesicle formation and stabilization in magnetotactic bacteria, providing numerous potential applications as nanoreactors for functional material synthesis, templating the fabrication of biocompatible functional one‐dimentional materials and drug carriers. Hirakawa and coworkers demonstrate that Pseudomonas putida P450 monooxygenase (P450s) is successfully activated by assembly with multiple molecules of auxiliary proteins, facilitating in vitro biotechnological application of P450s for chemical synthesis of valuable compounds. Wang et al describe a technique for tuning interparticle distance in gold nanoparticle by using the temperature‐dependent shrinkage/extension of DNA‐templated gold nanoparticle dimers, promising for application to the switching of optoelectronic nanodevices.…”