Understanding of real alternative redox partner of Streptomyces peucetius DoxA: Prediction and validation using in silico and in vitro analyses

Streptomyces peucetius ATCC27952 contains the cytochrome P450 monoxygenase DoxA that is responsible for the hydroxylation of daunorubicin into doxorubicin. Although S. peucetius ATCC27952 contains several potential redox partners, the most suitable endogenous electron-transport system is still unclear; therefore, we conducted a study of potential redox partners using Accelrys Discovery Studio 3.5. Recombinant DoxA along with its redox partners from S. peucetius FDX1, FDR2, and FDX3, and the putidaredoxin and putidaredoxin reductase from Pseudomonas putida that are essential equivalents of the class I type of bacterial electron-transport system were over-expressed and purified. The successful development of an efficient redox system was achieved by an in vitro enzymatic catalysis reaction with DoxA. The optimal pH for the activation of the heme was 7.6 and the optimal temperature was 30 degrees C. Our findings suggest a two-fold increase of DoxA activity via the NADH -> FDR2 -> FDX1 -> DoxA pathway for the hydroxylation of the daunorubicin, and indicate that the usage of a native redox partner may increase daunorubicin-derived doxorubicin production due to the inclusion of DoxA. (C) 2015 Elsevier Inc. All rights reserved.