A novel electron transport system for thermostable  CYP175A1 from Thermus thermophilus HB27.
CYP175A1 from Thermus thermophilus is a thermophilic cytochrome P450 and has great potential for industrial applications. However, a native electron transport system for CYP175A1 has not been identified. Here, an electron transport system for CYP175A1 was isolated from T. thermophilus HB27 by multistep chromatography, and identified as comprising ferredoxin (Fdx; locus in the genome, TTC1809) and ferredoxin-NAD(P)+reductase (FNR; locus in the genome, TTC0096) by N-terminal amino acid sequence analysis and MALDI-TOF-MS, respectively. Although TTC0096, which encodes the FNR, is annotated as a thioredoxin reductase in the T. thermophilus HB27 genome database, TTC0096 lacks an active-site dithiol/disulfide group, which is required to exchange reducing equivalents with thioredoxin. The FNR reduced ferricyanide, an artificial electron donor, in the presence of NADH and NADPH, but preferred NADPH as a cofactor (Km for NADH = 2440 +/- 546 microM; Km for NADPH = 4.1 +/- 0.2 microM). Furthermore, the FNR reduced cytochrome c in the presence of NADPH and Fdx. The Tm value of the FNR was 99 degrees C at pH 7.4. With an electron transport system consisting of Fdx and FNR, CYP175A1 efficiently catalyzed the hydroxylation of beta-carotene at the 3-position and 3'-position at 65 degrees C, and the Km and Vmax values for beta-carotene hydroxylation were 14.3 +/- 1.6 microM and 18.3 +/- 0.6 nmol beta-cryptoxanthin min(-1) nmol(-1) CYP175A1, respectively. This is the first report of a native electron transport system for CYP175A1.
Nanobiotechnology Research Center, Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, Gakuen, Sanda, Japan.
Takao
T
Mandai
Shinsuke
S
Fujiwara
Susumu
S
Imaoka
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000302:isolation & purification; D004579:Electron Transport; D005288:Ferredoxins / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D016415:Sequence Alignment; D013696:Temperature; D016963:Thermus thermophilus / Q000378:metabolism*; D019207:beta Carotene / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D005288:Ferredoxins; D019207:beta Carotene; D003577:Cytochrome P-450 Enzyme System