tailieunhanh - Báo cáo khoa học: A novel electron transport system for thermostable CYP175A1 from Thermus thermophilus HB27

CYP175A1 fromThermus thermophilusis a thermophilic cytochrome P450 and has great potential for industrial applications. However, a native elec-tron transport system for CYP175A1 has not been identified. Here, an elec-tron transport system for CYP175A1 was isolated from T. thermophilus HB27 by multistep chromatography, and identified as comprising ferre-doxin (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. . | ễFEBS Journal A novel electron transport system for thermostable CYP175A1 from Thermus thermophilus HB27 Takao Mandai Shinsuke Fujiwara and Susumu Imaoka Nanobiotechnology Research Center and Department of Bioscience Schoolof Science and Technology Kwansei Gakuin University Gakuen Sanda Japan Keywords CYP175A1 ferredoxin ferredoxin-NAD P reductase Thermus thermophilus p-carotene hydroxylase Correspondence S. Imaoka Department of Bioscience Schoolof Science and Technology Kwansei Gakuin University 2-1 Gakuen Sanda 669-1337 Japan Tel Fax 81 79 565 7673 E-mail imaoka@ Received 30 January 2009 revised 15 February 2009 accepted 18 February 2009 doi 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 M Km for NADPH 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 . With an electron transport system consisting of Fdx and FNR CYP175A1 efficiently catalyzed the hydroxylation of b-carotene at the 3-position and 3 -position at 65 C and the Km and Vmax values for b-carotene hydroxylation were M