Study on a Mechanism of Improving MaAPX1 Protein Activity by Mutating Methionine to Lysine

• Published in: Antioxidants Vol. 13; no. 7; p. 843
• Main Authors: Xiao, Lu, Jiang, Guoxiang, Lai, Hongmei, Duan, Xiaoyan, Yan, Huiling, Chen, Shaoge, Chen, Zexin, Duan, Xuewu
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 14-07-2024
• Subjects: activity; Antioxidants; ascorbate peroxidase 1 (APX1); Ascorbic acid; Bananas; Cell death; Cloning; E coli; Endangered & extinct species; Enzymatic activity; enzyme kinetics; Enzymes; Fruits; Gene expression; Genomes; Hydrogen peroxide; Lysine; Metabolism; Methionine; molecular docking; Mutagenesis; Mutants; Oxidative stress; Physiology; Potassium; Proteins; Reactive oxygen species; S-nitrosylation; Senescence; Spectroscopy; Brazil; China; Japan; Germany; molecular docking; activity; ascorbate peroxidase 1 (APX1); enzyme kinetics; S-nitrosylation
• ISSN: 2076-3921; 2076-3921
• DOI: 10.3390/antiox13070843

Ascorbate peroxidases (APXs) are key components of the ascorbate-glytathione cycle, which plays an important role in removing excess reactive oxygen species (ROS) in plants. Herein, MaAPX1 was verified as being involved in the ripening and senescence of banana fruit, exhibiting responsiveness to the accumulation of ROS and the oxidation of proteins. Site-directed mutation was applied to explore the mechanism of MaAPX1 activity changes. We found that the 32-site cysteine (Cys, C) served as a potential S-nitrosylation site. The mutant MaAPX1
activity was decreased significantly when Cys32 was mutated to serine (Ser, S). Intriguingly, the neighboring conserved 36-site methionine (Met, M), which is adjacent to Cys32, displayed an enzyme activity that was approximately five times higher than that of the wild-type MaAPX1 when mutated to lysine (Lys, K). Utilizing LC-MS/MS spectroscopy coupled with stopped-flow analysis showed that the enhanced MaAPX1
activity might be due to the increased S-nitrosylation level of Cys32 and the promotion of intermediate (compound
, the first intermediate product of the reaction of APX with H
O
) production. Molecular docking simulations showed that the S-N bond between Cys32 and Lys36 in MaAPX1
might have a function in protecting the thiol of Cys32 from oxidation. MaAPX1
, a promising mutant, possesses immense potential for improving the antioxidant capabilities of APX in the realm of bioengineering technology research.