Transition of stability of putidaredoxin reductase by introducing proline

Abstract

The monooxygenase activity of the Pseudomonas putida cytochrome P450 system was expressed in the presence of P450cam, putidaredoxin reductase (PdR), and putidaredoxin (Pd). In turn, it has been found to catalyze the oxidation of various organic compounds. Since cytochrome P450 system substrates are often insoluble in water, reactions should be carried out at high temperatures and in the presence of organic solvents where the high stability of all of P450cam, PdR, and Pd are needed. In this study, proline was introduced to improve the stability of PdR which was estimated to have the lowest organic solvent stability compared to others. Thus, the half-life of PdR_T221P at 40°C was 1.37 times longer than that of wild-type PdR. However, the half-life of PdR_T221P in the presence of 25% (v/v) methanol was shorter than that of wild-type PdR. Furthermore, structural stabilities of PdRs using molecular dynamics (MD) simulations demonstrated these phenomena.