A copper-zinc superoxide dismutase of Piper nigrum: Molecular cloning, recombinant expression, in vitro activity and molecular modeling

Abstract

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.