Metallothioneins (MTs) are low molecular weight, cysteine-rich, metal-binding proteins that are important for essential metal homeostasis, protection against oxidative stress, and buffering against toxic heavy metals. In this work the gene encoding an MT type 2 from Avicennia marina (Forssk.) Vierh. (AmMT2) was cloned into pET41a and transformed into the Escherichia coli strain Rosetta (DE3). Following the induction with isopropyl β-D-1-thiogalactopyranoside, AmMT2 was expressed as glutathione-S-transferase (GST)-tagged fusion protein. The accumulation of Zn2+, Cu2+, Fe2+, Ni2+ and Cd2+ for strain R-AmMT2 was 4, 8, 5.4, 2 and 1.6 fold of control strain suggesting the role of AmMT2 in accumulation of metals. Particularly the strain R-AmMT2 was able to accumulate 30.7 mg per g dry weight. The cells expressing AmMT2 was more tolerant to hydrogen peroxide and had higher catalase (CAT) activity. To understand the mechanistic action of AmMT2 hydrogen peroxide tolerance, the activity of CAT in the E. coli protein extract was assayed after addition of pure Fe2+/GST-AmMT complex and Apo/GST-AmMT2 in vitro. Whereas, the activity of CAT did not change by the addition of Apo/GST-AmMT2, the activity of CAT significantly increased after addition of Fe2+/GST-AmMT2. These results show that AmMT2 activates CAT through Fe2+ transfer which subsequently causes the oxidative stress tolerance.

中文翻译：

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来自 Avicennia marina 的 2 型金属硫蛋白与铁结合并通过激活过氧化氢酶来调节过氧化氢平衡

金属硫蛋白 (MTs) 是低分子量、富含半胱氨酸的金属结合蛋白，对基本金属稳态、防止氧化应激和缓冲有毒重金属很重要。在这项工作中，编码来自 Avicennia marina (Forssk.) Vierh 的 MT 类型 2 的基因。(AmMT2) 被克隆到 pET41a 中并转化到大肠杆菌菌株 Rosetta (DE3) 中。在用异丙基 β-D-1-硫代吡喃半乳糖苷诱导后，AmMT2 表达为谷胱甘肽-S-转移酶 (GST) 标记的融合蛋白。菌株 R-AmMT2 的 Zn2+、Cu2+、Fe2+、Ni2+ 和 Cd2+ 的积累是对照菌株的 4、8、5.4、2 和 1.6 倍，表明 AmMT2 在金属积累中的作用。特别是菌株 R-AmMT2 能够积累 30.7 mg/g 干重。表达 AmMT2 的细胞对过氧化氢的耐受性更高，并且具有更高的过氧化氢酶 (CAT) 活性。为了了解 AmMT2 过氧化氢耐受的机制作用，在体外添加纯 Fe2+/GST-AmMT 复合物和 Apo/GST-AmMT2 后，测定了大肠杆菌蛋白提取物中 CAT 的活性。然而，CAT的活性并没有因添加Apo/GST-AmMT2而改变，而添加Fe2+/GST-AmMT2后CAT的活性显着增加。这些结果表明，AmMT2 通过 Fe2+ 转移激活 CAT，从而导致氧化应激耐受。添加Apo/GST-AmMT2后CAT活性没有变化，添加Fe2+/GST-AmMT2后CAT活性显着增加。这些结果表明，AmMT2 通过 Fe2+ 转移激活 CAT，从而导致氧化应激耐受。添加Apo/GST-AmMT2后CAT活性没有变化，添加Fe2+/GST-AmMT2后CAT活性显着增加。这些结果表明，AmMT2 通过 Fe2+ 转移激活 CAT，从而导致氧化应激耐受。