TAILIEUCHUNG - Báo cáo Y học: Identification and characterization of thioredoxin and thioredoxin reductase fromAeropyrum pernixK1

We have identified and characterized a thermostable thio-redoxin system in the aerobic hyperthermophilic archaeon AeropyrumpernixK1. Thegene () of A. pernixencoding a 37 kDaprotein contains a redox active site motif (CPHC) but its N-terminal extension region (about 200 residues) shows no homologywithin the genome database. A second gene (Accession no. APE1061) has high homology to thioredoxin reductase and encodes a 37 kDa protein with the active site motif (CSVC), and binding sites forFADandNADPH | Eur. J. Biochem. 269 5423-5430 2002 FEBS 2002 doi Identification and characterization of thioredoxin and thioredoxin reductase from Aeropyrum pernix K1 Sung-Jong Jeon and Kazuhiko Ishikawa National Institute of Advanced Industrial Science and Technology Kansai Ikeda Osaka Japan We have identified and characterized a thermostable thioredoxin system in the aerobic hyperthermophilic archaeon Aeropyrumpernix K1. The gene Accession no. APE0641 of A. pernix encoding a 37 kDa protein contains a redox active site motif CPHC but its N-terminal extension region about 200 residues shows no homology within the genome database. A second gene Accession no. APE1061 has high homology to thioredoxin reductase and encodes a 37 kDa protein with the active site motif CSVC and binding sites for FAD and NADPH. We cloned the two genes and expressed both proteins in E. coli. It was observed that the recombinant proteins could act as an NADPH-dependent protein disulfide reductase system in the insulin reduction. In addition the APE0641 protein and thioredoxin reductase from E. coli could also catalyze the disulfide reduction. These indicated that APE1061 and APE0641 express thioredoxin ApTrx and thioredoxin reductase ApTR of A. pernix respectively. ApTR is expressed as an active homodimeric flavoprotein in the E. coli system. The optimum temperature was above 90 C and the half-life of heat inactivation was about 4 min at 110 C. The heat stability of ApTR was enhanced in the presence of excess FAD. ApTR could reduce both thioredoxins from A. pernix and E. coli and showed a similar molar specific activity for both proteins. The standard state redox potential of ApTrx was about -262 mV which was slightly higher than that of Trx from E. coli -270 mV . These eesults indicate that a lower redox potential of thioredoxin is not necessary for keeping catalytic disulfide bonds reduced and thereby coping with oxidative stress in an aerobic hyperthermophilic archaea. .

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