novel electron transport system for thermostable CYP175A1 from Thermus thermophilus HB27

• Published in: The FEBS journal 2009-04, Vol.276 (8), p.2416-2429
• Main Authors: Mandai, Takao, Fujiwara, Shinsuke, Imaoka, Susumu
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; beta Carotene - chemistry; beta Carotene - metabolism; Biochemistry; Chromatography; CYP175A1; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - isolation & purification; Electron Transport; Electrons; ferredoxin; ferredoxin-NAD(P)⁺ reductase; Ferredoxins - chemistry; Ferredoxins - metabolism; ferredoxin–NAD(P)+ reductase; Genomics; Hydrogen-Ion Concentration; Hydrolysis; Molecular Sequence Data; Mutagenesis, Site-Directed; Phylogeny; Sequence Alignment; Temperature; Thermus thermophilus; Thermus thermophilus - metabolism; β-carotene hydroxylase
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/j.1742-4658.2009.06974.x

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 μ m; Km for NADPH = 4.1 ± 0.2 μ m). Furthermore, the FNR reduced cytochrome c in the presence of NADPH and Fdx. The Tm value of the FNR was 99 °C at pH 7.4. With an electron transport system consisting of Fdx and FNR, CYP175A1 efficiently catalyzed the hydroxylation of β-carotene at the 3-position and 3'-position at 65 °C, and the Km and Vmax values for β-carotene hydroxylation were 14.3 ± 1.6 μ m and 18.3 ± 0.6 nmol β-cryptoxanthin·min⁻¹·nmol⁻¹ CYP175A1, respectively. This is the first report of a native electron transport system for CYP175A1.