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 °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.
Department of Pharmaceutical Engineering, SunMoon University, #100, Kalsan-ri, Tangjeong-myeon, Asansi, Chungnam 336-708, Republic of Korea.
Hemraj
H
Rimal
Department of Pharmaceutical Engineering, SunMoon University, #100, Kalsan-ri, Tangjeong-myeon, Asansi, Chungnam 336-708, Republic of Korea.
Seung-Won
SW
Lee
Department of Pharmaceutical Engineering, SunMoon University, #100, Kalsan-ri, Tangjeong-myeon, Asansi, Chungnam 336-708, Republic of Korea.
Joo-Ho
JH
Lee
Department of Pharmaceutical Engineering, SunMoon University, #100, Kalsan-ri, Tangjeong-myeon, Asansi, Chungnam 336-708, Republic of Korea. Electronic address: tjoh3782@sunmoon.ac.kr.
Tae-Jin
TJ
Oh
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics; D003630:Daunorubicin / Q000737:chemistry*; D004317:Doxorubicin / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005288:Ferredoxins / Q000737:chemistry*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D006900:Hydroxylation; D007700:Kinetics; D062105:Molecular Docking Simulation; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016958:Pseudomonas putida / Q000737:chemistry / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013302:Streptomyces / Q000737:chemistry* / Q000201:enzymology; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D005288:Ferredoxins; D011994:Recombinant Proteins; C100137:putidaredoxin; D004317:Doxorubicin; D003577:Cytochrome P-450 Enzyme System; C116920:doxA protein, Streptomyces peucetius; D009247:NADH, NADPH Oxidoreductases; C022486:putidaredoxin reductase; D003630:Daunorubicin
Cytochrome P450; Electron-transport system; Homology modeling; In silico analysis; Streptomyces peucetius ATCC27952