Functional Analysis of Free Methionine-R-sulfoxide Reductase from Saccharomyces cerevisiae
Functional Analysis of Free Methionine-R-sulfoxide Reductase from Saccharomyces cerevisiae
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DOI：
10.1074/jbc.m805891200
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发表时间：
2009-02-13
影响因子：
4.8
通讯作者：
Gladyshev, Vadim N.
中科院分区：
文献类型：
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作者：
Le, Dung Tien;Lee, Byung Cheon;Gladyshev, Vadim N.
蛋氨酸亚砜还原酶（Msrs）是一种氧化还原酶，可催化氧化型蛋氨酸的巯基依赖性还原反应。MsrA和MsrB是最为人熟知的Msrs，它们分别修复蛋白质中的蛋氨酸 - S - 亚砜（Met - S - SO）和蛋氨酸 - R - 亚砜（Met - R - SO）残基。此外，最近发现了一种对游离Met - R - SO具有特异性的大肠杆菌酶，被命名为fRMsr。在这项工作中，我们进行了比较基因组学和实验分析，以研究fRMsr的存在、进化和功能。这种蛋白质在大约一半的原核生物和单细胞真核生物中以单拷贝和两种相互排斥的亚型存在，但在高等植物和动物中缺失。发现酿酒酵母的一种fRMsr同源物可还原游离的Met - R - SO，但不能还原游离的Met - S - SO或丹磺酰 - Met - R - SO。fRMsr负责酵母细胞在Met - R - SO上的生长，并且fRMsr/MsrA双突变体在蛋氨酸亚砜混合物上无法生长。然而，在有蛋氨酸存在的情况下，即使是fRMsr/MsrA/MsrB三突变体也是有活力的。此外，fRMsr缺失菌株对氧化应激的敏感性增加，寿命缩短，而fRMsr的过表达赋予了对氧化剂更高的抗性。分子建模以及硫氧还蛋白对半胱氨酸残基的靶向作用表明，在酵母fRMsr中，Cys(101)是催化残基，Cys(125)是分解残基。这些残基以及第三个半胱氨酸（分解Cys(91)）在结构上聚集在一起，并且每个残基都是该酶催化活性所必需的。数据表明，fRMsr是负责酿酒酵母中游离Met - R - SO还原的主要酶。
Methionine sulfoxide reductases (Msrs) are oxidoreductases that catalyze thiol-dependent reduction of oxidized methionines. MsrA and MsrB are the best known Msrs that repair methionine-S-sulfoxide (Met-S-SO) and methionine-R-sulfoxide (Met-R-SO) residues in proteins, respectively. In addition, an Escherichia coli enzyme specific for free Met-R-SO, designated fRMsr, was recently discovered. In this work, we carried out comparative genomic and experimental analyses to examine occurrence, evolution, and function of fRMsr. This protein is present in single copies and two mutually exclusive subtypes in about half of prokaryotes and unicellular eukaryotes but is missing in higher plants and animals. A Saccharomyces cerevisiae fRMsr homolog was found to reduce free Met-R-SO but not free Met-S-SO or dabsyl-Met-R-SO. fRMsr was responsible for growth of yeast cells on Met-R-SO, and the double fRMsr/MsrA mutant could not grow on a mixture of methionine sulfoxides. However, in the presence of methionine, even the triple fRMsr/MsrA/MsrB mutant was viable. In addition, fRMsr deletion strain showed an increased sensitivity to oxidative stress and a decreased life span, whereas overexpression of fRMsr conferred higher resistance to oxidants. Molecular modeling and cysteine residue targeting by thioredoxin pointed to Cys(101) as catalytic and Cys(125) as resolving residues in yeast fRMsr. These residues as well as a third Cys, resolving Cys(91), clustered in the structure, and each was required for the catalytic activity of the enzyme. The data show that fRMsr is the main enzyme responsible for the reduction of free Met-R-SO in S. cerevisiae.