Xiao Lu, Jiang Guoxiang, Lai Hongmei, Duan Xiaoyan, Yan Huiling, Chen Shaoge, Chen Zexin, Duan Xuewu
Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China.
Institute of Quality Standard and Monitoring Technology for Agro-Products of Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China.
Antioxidants (Basel). 2024 Jul 14;13(7):843. 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 HO) 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.
抗坏血酸过氧化物酶（APXs）是抗坏血酸-谷胱甘肽循环的关键组成部分，该循环在清除植物体内过量的活性氧（ROS）方面发挥着重要作用。在此，MaAPX1被证实参与香蕉果实的成熟和衰老过程，对ROS的积累和蛋白质氧化具有响应性。应用定点突变来探究MaAPX1活性变化的机制。我们发现32位的半胱氨酸（Cys，C）作为一个潜在的S-亚硝基化位点。当Cys32突变为丝氨酸（Ser，S）时，突变型MaAPX1的活性显著降低。有趣的是，与Cys32相邻的保守36位甲硫氨酸（Met，M），在突变为赖氨酸（Lys，K）时，其酶活性比野生型MaAPX1高出约五倍。利用液相色谱-串联质谱联用技术结合停流分析表明，MaAPX1活性增强可能是由于Cys32的S-亚硝基化水平增加以及中间产物（化合物I，APX与HO反应的第一个中间产物）生成的促进。分子对接模拟表明，MaAPX1中Cys32和Lys36之间的S-N键可能具有保护Cys32的巯基不被氧化的功能。MaAPX1作为一个有前景的突变体，在生物工程技术研究领域具有提高APX抗氧化能力的巨大潜力。
