Dual-domain superoxide dismutase: In silico prediction directed combinatorial mutation for enhanced robustness and...

The robustness and catalytic activity of superoxide dismutase (SOD) are still the main factors limiting their application in industrial fields. This study aims to further improve the properties of a natural thermophilic iron/manganese dual-domain SOD (Fe/Mn-SODA fused with N-terminal polypeptide) from Geobacillus thermodenitrificans NG80-2 (GtSOD) by modifying its each domain using in-depth in silico prediction analysis as well as protein engineering. First, computational analysis of the N-terminal domain and GtSODA domain was respectively performed by using homologous sequence alignment and virtual mutagenesis. Seven proposed mutation sites favoring increased robustness were screened out for single-point mutants (SPMs) construction. Enzymatic characterization of these SPMs identified the most favorable mutation sites E107 and S265 located in two different domains. Subsequently, the dual-domain site combinatorial mutant (DDSCM) E107L/S265K showed significant superposition effects and additional improvement in catalytic efficiency, with a Kcat/Km value of 145.45 %, 33.66 %, and 60.33 % higher than the wild type (WT), the SPMs E107L and S265K, respectively. Molecular dynamics simulations, structural and surface charge analysis revealed the possible mechanism by which combinatorial mutations improve the robustness and catalytic activity of GtSOD. Furthermore, DDSCM showed more significant resistance to ultraviolet B and various stress than WT, indicating its highly competitive industrial application prospects.