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

Jingjing Liu; Lei Yao; Fang Zhao; Jingjing Zhang; Jingbo Gong; Jiabin Li; Ya Bian; Yalin Yin; Rui Zhao; Yijia Wang; Wei Wang

doi:10.1016/j.ijbiomac.2024.139179

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

Int J Biol Macromol. 2024 Dec 26:292:139179. doi: 10.1016/j.ijbiomac.2024.139179. Online ahead of print.

Authors

Jingjing Liu¹, Lei Yao¹, Fang Zhao¹, Jingjing Zhang¹, Jingbo Gong¹, Jiabin Li¹, Ya Bian¹, Yalin Yin¹, Rui Zhao¹, Yijia Wang², Wei Wang³

Affiliations

¹ Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, TEDA Institute of Biological Sciences and Biotechnology, Nankai University, TEDA, Tianjin 300457, PR China.
² Laboratory of Oncologic Molecular Medicine, Tianjin Union Medical Center, Nankai University, Tianjin 300121, PR China.
³ Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, TEDA Institute of Biological Sciences and Biotechnology, Nankai University, TEDA, Tianjin 300457, PR China; Tianjin Key Laboratory of Microbial Functional Genomics, Tianjin 300457, PR China. Electronic address: nkweiwang@nankai.edu.cn.

PMID: 39732240
DOI: 10.1016/j.ijbiomac.2024.139179

Abstract

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.

Keywords: Catalytic activity; Combinatorial mutation; Geobacillus thermodenitrificans; Multiple domains; Robustness; Superoxide dismutase.