Tài liệu Báo cáo khoa học: A novel electron transport system for thermostable CYP175A1 from Thermus

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, School of Science and Technology, Kwansei Gakuin University,

Gakuen, Sanda, Japan

Cytochrome P450s are associated with a number of

physiologically essential reactions, including drug

metabolism, carbon source assimilation, and the bio￾synthesis of steroids, vitamins, prostaglandins, and

antibiotics [1]. Cytochrome P450s have great potential

to perform numerous industrially important reactions.

Indeed, cytochrome P450sca-2 from Streptomyces car￾bophilus has already been used for the production of

pravastatin, a cholesterol-lowering drug [2]. However,

low tolerance to various solvents and high temperature

has generally limited the usefulness of cyto￾chrome P450s for industrial applications. Thermophilic

cytochrome P450s possess extreme stability, and

might be used to overcome such limitations. Recently,

two thermophilic cytochrome P450s, CYP119 and

CYP175A1, were identified in Sulfolobus solfataricus

and Thermus thermophilus, respectively [3,4].

CYP119 is well characterized, and its crystal struc￾ture has been determined in the ligand-free state and

in several ligand-bound states [5,6]. As expected,

CYP119 is highly resistant to both high temperatures

(Tm = 91 C) and high pressures (up to 2 kbar) [7].

Keywords

CYP175A1; ferredoxin; ferredoxin–NAD(P)+

reductase; Thermus thermophilus;

b-carotene hydroxylase

Correspondence

S. Imaoka, Department of Bioscience,

School of Science and Technology, Kwansei

Gakuin University, 2-1 Gakuen, Sanda

669-1337, Japan

Tel ⁄ Fax: +81 79 565 7673

E-mail: [email protected]

(Received 30 January 2009, revised 15

February 2009, accepted 18 February 2009)

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 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. Although TTC0096,

which encodes the FNR, is annotated as a thioredoxin reductase in the

T. thermophilus HB27 genome database, TTC0096 lacks an active-site dithi￾ol ⁄ 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 lm; Km for NADPH = 4.1 ± 0.2 lm).

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 cata￾lyzed 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

14.3 ± 1.6 lm and 18.3 ± 0.6 nmol b-cryptoxanthinÆmin)1

Ænmol)1

CYP175A1, respectively. This is the first report of a native electron trans￾port system for CYP175A1.

Abbreviations

Fdx, ferredoxin; FNR, ferredoxin–NAD(P)+ reductase; IPTG, isopropyl-thio-b-D-galactoside; OFOR, 2-oxoacid:ferredoxin oxidoreductase;

ONFR, oxygenase-coupled NADH–ferredoxin reductase; SD, standard deviation; TR, thioredoxin reductase; UPLC, ultra-performance liquid

chromatography.

2416 FEBS Journal 276 (2009) 2416–2429 ª 2009 The Authors Journal compilation ª 2009 FEBS