Superoxide dismutase (SOD) plays crucial role controlling oxidative burst produced during interactions of plants with biotic factors. Previous studies identified a partial cDNA sequence coding for SOD in black pepper (Piper nigrum L.) infected by Fusarium solani f. sp. piperis, causal agent of rot root disease. Here, our main aim was to isolate this full-length cDNA sequence and characterize the P. nigrum SOD, named PnCuZnSOD. The 738-bp full-length cDNA contains a 459-bp open reading frame (ORF) encoding a deduced protein with 152 amino acid residues, predicted to be a cytosolic protein with molecular weight and isoelectric point of 15 kDa and 5.27, respectively. Comparative sequence analysis revealed high identity between the deduced PnCuZnSOD and superoxide dismutase sequences from GenBank database. In addition, putative conserved motifs, copper- and zinc-binding domains and cysteine residues involved in disulfide bond were identified. Furthermore, molecular modeling analyzes generated the three-dimensional structure of PnCuZnSOD protein. The recombinant PnCuZnSOD was produced by heterologous expression in bacterial cells, followed by the His-tagged protein purification and enzymatic activity evaluation. The effects of heat and hydrogen peroxide on the PnCuZnSOD activity were evaluated. The results indicated PnCuZnSOD as a probable active protein contributing to reactive oxygen species (ROS) homeostasis in the cytosol of P. nigrum cells.

中文翻译：

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Piper nigrum 的一种铜-锌超氧化物歧化酶：分子克隆、重组表达、体外活性和分子模型

超氧化物歧化酶 (SOD) 在控制植物与生物因子相互作用过程中产生的氧化爆发中起着至关重要的作用。先前的研究确定了在被茄病镰刀菌感染的黑胡椒（Piper nigrum L.）中编码 SOD 的部分 cDNA 序列。sp。胡椒，腐烂根病的病原体。在这里，我们的主要目的是分离这个全长 cDNA 序列并表征 P. nigrum SOD，命名为 PnCuZnSOD。738-bp 全长 cDNA 包含一个 459-bp 的开放阅读框 (ORF)，编码具有 152 个氨基酸残基的推断蛋白质，预测为分子量和等电点分别为 15 kDa 和 5.27 的胞质蛋白。比较序列分析显示，推断的 PnCuZnSOD 与 GenBank 数据库中的超氧化物歧化酶序列具有高度同一性。此外，推定的保守基序，鉴定了铜和锌结合结构域以及参与二硫键的半胱氨酸残基。此外，分子建模分析产生了 PnCuZnSOD 蛋白的三维结构。重组 PnCuZnSOD 是通过在细菌细胞中异源表达产生的，然后进行 His 标签蛋白纯化和酶活性评估。评估了热和过氧化氢对 PnCuZnSOD 活性的影响。结果表明，PnCuZnSOD 可能是一种活性蛋白，有助于 P. nigrum 细胞胞质溶胶中的活性氧 (ROS) 稳态。重组 PnCuZnSOD 是通过在细菌细胞中异源表达产生的，然后进行 His 标签蛋白纯化和酶活性评估。评估了热和过氧化氢对 PnCuZnSOD 活性的影响。结果表明，PnCuZnSOD 可能是一种活性蛋白，有助于 P. nigrum 细胞胞质溶胶中的活性氧 (ROS) 稳态。重组 PnCuZnSOD 是通过在细菌细胞中异源表达产生的，然后进行 His 标签蛋白纯化和酶活性评估。评估了热和过氧化氢对 PnCuZnSOD 活性的影响。结果表明，PnCuZnSOD 可能是一种活性蛋白，有助于 P. nigrum 细胞胞质溶胶中的活性氧 (ROS) 稳态。