PDF(Pubmed)
Abstract：
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 MaAPX1C32S 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 MaAPX1M36K activity might be due to the increased S-nitrosylation level of Cys32 and the promotion of intermediate (compound I, the first intermediate product of the reaction of APX with H2O2) production. Molecular docking simulations showed that the S-N bond between Cys32 and Lys36 in MaAPX1M36K might have a function in protecting the thiol of Cys32 from oxidation. MaAPX1M36K, a promising mutant, possesses immense potential for improving the antioxidant capabilities of APX in the realm of bioengineering technology research.
摘要：
抗坏血酸过氧化物酶（APXs）是抗坏血酸-谷胱甘肽循环的关键成分，在去除植物中过量的活性氧（ROS）中起着重要作用。在这里，MaAPX1被证实参与了香蕉果实的成熟和衰老，对ROS的积累和蛋白质的氧化表现出响应性。采用定点突变方法探讨MaAPX1活性变化的机制。我们发现32位半胱氨酸（Cys，C)充当潜在的S-亚硝基化位点。当Cys32突变为丝氨酸时，突变体MaAPX1C32S活性显着降低（Ser，S).有趣的是，邻近的保守的36位点蛋氨酸(Met，M),当突变为赖氨酸时，与Cys32相邻的酶活性比野生型MaAPX1高约五倍（Lys，K).利用LC-MS/MS光谱结合停流分析表明，增强的MaAPX1M36K活性可能是由于Cys32的S-亚硝基化水平增加和中间体的促进（化合物I，APX与H2O2）生产反应的第一个中间产物。分子对接模拟显示MaAPX1M36K中Cys32和Lys36之间的S-N键可能具有保护Cys32的硫醇免于氧化的功能。MaAPX1M36K,一个有前途的变种人,在生物工程技术研究领域具有巨大的提高APX抗氧化能力的潜力。
