School of Biological Sciences, Flinders University, GPO Box 2100, Adelaide SA5001, Australia.
Comp Biochem Physiol B Biochem Mol Biol. 2010 Jun;156(2):97-106. doi: 10.1016/j.cbpb.2010.02.011. Epub 2010 Mar 6.
Peroxiredoxins (Prxs, EC: 1.11.1.15) are cysteine-dependent peroxidases proposed to function as antioxidant enzymes and also in H2O2-mediated cell signaling. They have been well characterized in yeast, mammals, protists and bacteria but not yet in fish. Here we describe the cloning and functional characterization of a Prx 2 cDNA from southern bluefin tuna (SBT, Thunnus maccoyii), an important aquaculture species in South Australia. The SBT Prx sequence was closely related (76-92% identical) to Prx 1 and 2 sequences from other fish and mammals and phylogenetic analyses showed that it was most likely a Prx 2. The deduced amino acid sequence contained the peroxidatic and resolving Cys residues characteristic of typical 2-Cys Prx proteins from all kingdoms of life. It also contained the GGLG motif associated with the sensitivity of eukaryotic typical 2-Cys Prx proteins to overoxidation and consequent inactivation by H2O2. When the SBT Prx 2 was expressed in E. coli, it showed thioredoxin (Trx)-dependent peroxidase activity with H2O2, cumene hydroperoxide (CuOOH) and t-butyl hydroperoxide (t-bOOH). The SBT Prx displayed Michaelis-Menten kinetics with Trx but sigmoidal kinetics with H2O2 and CuOOH. The K(m)(Trx) was 12 microM and the S(0.5) values for H2O2 and CuOOH were 29 and 25 microM, respectively. At mM concentrations of H2O2, SBT Prx progressively lost its peroxidase activity as has been observed for other eukaryotic typical 2-Cys Prx proteins. The native SBT Prx enzyme existed as a mixture of dimers, tetramers, decamers and a higher order aggregate.
过氧化物酶（Prxs，EC：1.11.1.15）是一类依赖半胱氨酸的过氧化物酶，被认为具有抗氧化酶的功能，并且在 H2O2 介导的细胞信号转导中发挥作用。它们在酵母、哺乳动物、原生动物和细菌中得到了很好的描述，但在鱼类中尚未得到描述。在这里，我们描述了南方蓝鳍金枪鱼（SBT，Thunnus maccoyii）Prx 2 cDNA 的克隆和功能特征，南方蓝鳍金枪鱼是南澳大利亚重要的水产养殖物种。SBT Prx 序列与来自其他鱼类和哺乳动物的 Prx 1 和 2 序列密切相关（76-92%相同），系统发育分析表明它很可能是 Prx 2。推导的氨基酸序列包含所有生命领域典型 2-Cys Prx 蛋白的过氧化物酶和解析 Cys 残基。它还包含与真核生物典型 2-Cys Prx 蛋白对过氧化物的敏感性以及 H2O2 引起的失活相关的 GGLG 基序。当 SBT Prx 2 在大肠杆菌中表达时，它显示出依赖硫氧还蛋白（Trx）的过氧化物酶活性，具有 H2O2、cumene hydroperoxide（CuOOH）和 t-butyl hydroperoxide（t-bOOH）。SBT Prx 与 Trx 表现出米氏动力学，与 H2O2 和 CuOOH 表现出 S 形动力学。K(m)(Trx)为 12 microM，H2O2 和 CuOOH 的 S(0.5)值分别为 29 和 25 microM。在 H2O2 的 mM 浓度下，SBT Prx 逐渐失去其过氧化物酶活性，这在其他真核典型 2-Cys Prx 蛋白中也观察到。天然的 SBT Prx 酶以二聚体、四聚体、十聚体和更高阶聚合体的混合物形式存在。
