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首页> 外文期刊>Journal of bacteriology >Characterization of a Thioredoxin-Thioredoxin Reductase System from the Hyperthermophilic Bacterium Thermotoga maritima
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Characterization of a Thioredoxin-Thioredoxin Reductase System from the Hyperthermophilic Bacterium Thermotoga maritima

机译：嗜热菌嗜热菌中硫氧还蛋白-硫氧还蛋白还原酶系统的表征

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A thioredoxin reductase and a thioredoxin were purified to homogeneity from a cell extract of Thermotoga maritima. The thioredoxin reductase was a homodimeric flavin adenine dinucleotide (FAD)-containing protein with a subunit of 37 kDa estimated using SDS-PAGE, which was identified to be TM0869. The amino acid sequence of the enzyme showed high identities and similarities to those of typical bacterial thioredoxin reductases. Although the purified T. maritima thioredoxin reductase could not use thioredoxin from Spirulina as an electron acceptor, it used thioredoxin that was purified from T. maritima by monitoring the dithiothreitol-dependent reduction of bovine insulin. This enzyme also catalyzed the reduction of benzyl viologen using NADH or NADPH as an electron donor with apparent Vmax values of 1,111 ± 35 μmol NADH oxidized min?1mg?1 and 115 ± 2.4 μmol NADPH oxidized min?1mg?1, respectively. The apparent Km values were determined to be 89 ± 1.1 μM, 73 ± 1.6 μM, and 780 ± 20 μM for benzyl viologen, NADH, and NADPH, respectively. Optimal pH values were determined to be 9.5 and 6.5 for NADH and NADPH, respectively. The enzyme activity increased along with the rise of temperature up to 95°C, and more than 60% of the activity remained after incubation for 28 h at 80°C. The purified T. maritima thioredoxin was a monomer with a molecular mass of 31 kDa estimated using SDS-PAGE and identified as TM0868, which exhibited both thioredoxin and thioltransferase activities. T. maritima thioredoxin and thioredoxin reductase together were able to reduce insulin or 5,5′-dithio-bis(2-nitrobenzoic acid) using NAD(P)H as an electron donor. This is the first thioredoxin-thioredoxin reductase system characterized from hyperthermophilic bacteria.
机译：从 Thermotoga maritima 的细胞提取物中纯化出硫氧还蛋白还原酶和硫氧还蛋白。硫氧还蛋白还原酶是含同二聚体黄素腺嘌呤二核苷酸（FAD）的蛋白，使用SDS-PAGE估计其亚基为37 kDa，鉴定为TM0869。该酶的氨基酸序列与典型的细菌硫氧还蛋白还原酶具有高度同一性和相似性。尽管纯化的 T。 maritima 硫氧还蛋白还原酶不能使用 Spirulina 的硫氧还蛋白作为电子受体，而是使用从 T纯化的硫氧还蛋白。监测二硫苏糖醇依赖性减少牛胰岛素的作用。该酶还使用NADH或NADPH作为电子供体，催化苄基紫精还原，表观 V max 值为1,111±35μmolNADH氧化min ？1 < / sup> mg ？1 和115±2.4μmolNADPH氧化min ？1 mg ？1 。苄基紫精，NADH和NADPH的表观 K m 值分别确定为89±1.1μM，73±1.6μM和780±20μM。对于NADH和NADPH，最佳pH值分别确定为9.5和6.5。酶的活性随着温度的升高而升高，最高温度可达95°C，在80°C孵育28小时后，酶的活性仍超过60％。纯化的 T。 maritima硫氧还蛋白是通过SDS-PAGE估计分子量为31 kDa的单体，鉴定为TM0868，具有硫氧还蛋白和硫醇转移酶的活性。 T。 maritima硫氧还蛋白和硫氧还蛋白还原酶一起能够使用NAD（P）H作为电子供体来还原胰岛素或5,5'-二硫代双（2-硝基苯甲酸）。这是第一个以超嗜热细菌为特征的硫氧还蛋白-硫氧还蛋白还原酶系统。

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