酵母谷胱甘肽转移酶Gtt2和甲硫氨酸亚砜还原酶Mxr1的结构与功能研究
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摘要
(Ⅰ)自然界中有一类能够损伤细胞或者破坏遗传物质的有毒物质,叫异生质(xenobiotics)。它们或者自生物体外进入,或者由体内代谢产生。为了消除这些有毒的异生质,生物体在漫长的进化过程中产生了一系列成熟有效的解毒机制。总的来说,这些解毒过程可以分为三个流程:一,通过引入活性功能基团激活化学性质相对稳定的异生质;二,将异生质上的活性功能基团与其他物质偶联,生成新的无毒或弱毒物质;三,偶联后的异生质溶解度大幅上升,可以通过膜表面的通道排出细胞,或者被下游反应最终降解。
谷胱甘肽转移酶(glutathione S-transferase,GST,EC 2.5.1.18)就是在解毒第二阶段中发挥重要作用的一种酶。该家族的酶能够催化异生质与谷胱甘肽(glutathione,GSH)巯基的偶联,进而消除异生质的亲电基团,降低毒性,提高溶解度。真核生物通常含有多个直系同源的谷胱甘肽转移酶,参与不同的反应流程。在哺乳动物中,胞质内的谷胱甘肽转移酶全都是以二体形式存在,并根据一级序列被划分为八个主要亚家族:Alpha、Kappa、Pi、Mu、Theta、Zeta、Sigma和Omega。在对非哺乳动物的研究中,一些新的谷胱甘肽转移酶亚家族被报道,如细菌内的Beta亚家族,高等植物中的Phi和Tau亚家族,昆虫中的Delta亚家族。真菌也含有多种谷胱甘肽转移酶,但是一级序列变化较大,目前无法归入依照序列划分的分类体系。除了这种分类体系,还有一种根据谷胱甘肽转移酶催化残基的分类方法。谷胱甘肽转移酶在催化过程中,将依靠某一特定催化残基去降低谷胱甘肽的pKa值,通过氢键稳定巯基负离子,从而加速异生质的亲电攻击。该分类方法依照反应过程中催化残基的类型,将所有的胞内谷胱甘肽转移酶分为三大类:酪氨酸催化(Tyr-),丝氨酸催化(Ser-),半胱氨酸催化(Cys-)。目前所有的胞内谷胱甘肽转移酶都可以归入上述三个大类。据报道,催化残基的突变可以显著降低,甚至完全消除谷胱甘肽转移酶的活性。
在酿酒酵母(Saccharomyces cerevisiae)中,目前发现七种蛋白具有谷胱甘肽转移酶活性,分别是Gtt1、Gtt2、Gto1、Gto2、Gto3、Grx1和Grx2。从已知的结构看,Grx1和Grx2并不属于谷胱甘肽转移酶家族,因为它们没有谷胱甘肽转移酶的C端结构域,而只具有N端结构域。在剩下的五个蛋白均属于谷胱甘肽转移酶家族,其中Gto1/2/3被归入谷胱甘肽转移酶Omega亚家族,Gtt1和Gtt2因序列同源度过低而无法归入现有的亚家族。虽然这五个谷胱甘肽转移酶的生理和生化功能都被广泛研究,但是到目前为止它们的三维结构还没有被报道。因此,我们展开了一项关于酿酒酵母谷胱甘肽转移酶三维结构的研究。我们解析了酿酒酵母中谷胱甘肽转移酶Gtt2的结构(2.23 (A|°)),以及Gtt2分别与底物谷胱甘肽GSH(2.10 (A|°))和底物类似物磺化谷胱甘肽GTS(2.20 (A|°))的复合物结构。从整体结构上看,Gtt2与常见的谷胱甘肽转移酶没有太大区别。但是通过对催化位点的结构分析、定点突变、光谱学分析和酶活测定,我们发现Gtt2与前人报道的三种催化类型(Tyr-、Ser-、Cys-)显著不同。Gtt2是通过C端结构域上的一个丝氨酸和一个组氨酸共同稳定的水分子来发挥催化功能的,而不是通过某个催化残基上的极性原子。另外,同源序列比对、定点突变与酶活测定证明,伸向催化位点的α1螺旋的N端,由于空间位阻的原因,只能是甘氨酸或者丙氨酸这两种侧链较小的残基。最终,我们定义了一类新的谷胱甘肽转移酶催化类型:非典型性(Atypical-type)谷胱甘肽转移酶。
(Ⅱ)硫氧还蛋白(thioredoxin,Trx)是一类广泛存在于自然界中的“巯基—二硫键”交换蛋白。它参与了很多重要的胞内反应,比如还原甲硫氨酸亚砜、核糖核苷酸、过氧化物等等。硫氧还蛋白家族都具有一个高度保守的CXXC活性相关结构域。反应过程中,第一个半胱氨酸会攻击底物蛋白的分子内二硫键,将分子内二硫键转化成分子间二硫键,形成反应中间态。此时硫氧还蛋白和底物蛋白会形成一个极不稳定的二硫键连接的复合物。之后,该分子间二硫键会被硫氧还蛋白CXXC中的第二个Cys攻击,中间态复合物解体,释放出被还原了的底物蛋白和被氧化了的硫氧还蛋白。
甲硫氨酸是一种对氧化非常敏感的氨基酸。在活性氧簇或活性氮簇存在时,甲硫氨酸很容易被氧化成甲硫氨酸亚砜(Met-SO)。由于是手性分子,所以氧化产物是以镜像对映体的形式(Met-S-SO and Met-R-SO)混合存在。蛋白表面的甲硫氨酸被氧化后会对细胞造成一系列损伤并最终加速衰老过程。然而,细胞内有一种称为甲硫氨酸亚砜还原酶(methionine-S-sulfoxide reductase,Msr)的蛋白,能够用硫氧还蛋白Trx提供的电子,去还原被氧化生成的甲硫氨酸亚砜,重新变成甲硫氨酸。最近,法国一科研小组报道,不光是硫氧还蛋白可以给甲硫氨酸亚砜还原酶提供电子,谷氧还蛋白/谷胱甘肽系统同样可以作为还原力的提供者。不论其还原力来源于哪里,甲硫氨酸亚砜还原酶都被证明在酿酒酵母、昆虫、哺乳动物体内,有延长寿命的作用。
到目前为止,一共有三个甲硫氨酸亚砜还原酶家族被报道(MsrA、MsrB、fRMsr)。MsrA和MsrB是最先被发现的两个家族,对底物有手性要求,分别能还原Met-S-SO和Met-R-SO。他们不但能够还原游离状态的甲硫氨酸亚砜,还能够还原蛋白表面的甲硫氨酸亚砜。据报道,一系列与人类疾病相关的蛋白都被证明是MsrA和MsrB的底物,比如钙调蛋白、HIV-2蛋白酶、Alpha-1蛋白酶抑制剂等等。fRMsr家族直到最近才被发现,它的特点是只能够还原游离状态的Met-R-SO,对蛋白表面的Met-R-SO则无能为力。尽管这三个家族有不同的起源、结构、底物特异性和物种分布,但它们所涉及的催化机理却是基本一致的。都是甲硫氨酸亚砜被还原并释放,催化半胱氨酸的巯基氧化成次磺酸,转化为分子内二硫键,最后由硫氧还蛋白或者其他电子供体还原Msr的分子内二硫键。
现在已经有七个不同物种的MsrA结构被报道。不论是X射线晶体衍射方法解析的还是核磁共振波谱法解析的,它们的核心结构域基本一致,可以很好的叠合。各结构之间的区别主要集中在活性位点周围的柔性区,尤其是C末端的柔性区,这恰恰是参与催化的一个半胱氨酸所在的位置,暗示这些柔性区的构象变化很可能会促进分子内二硫键的形成,并与硫氧还蛋白相互作用有关。
酿酒酵母(Saccharomyces cerevisiae)中存在三个甲硫氨酸亚砜还原酶,分别是Mxr1/MsrA、Mxr2/MsrB和Ykg9/fRMsr。尽管这三个甲硫氨酸亚砜还原酶的机制已经被广泛研究,但是它们为什么能够与多种的底物蛋白或者硫氧还蛋白相互作用,仍然还不清楚。为此,我们解析了Mxr1还原态(2.04 (A|°)),二体氧化态(1.90 (A|°))以及与硫氧还蛋白Trx2结合的复合物(2.70 (A|°))的晶体结构。通过结构叠合分析,我们发现活性位点周围的三段柔性区构象变化很大,可以根据底物蛋白的表面而改变构象,这很有可能就是甲硫氨酸亚砜还原酶底物多样性的原因。另外,该区域与Trx2相互作用时所显示的柔性,提供了Trx能用单一活性位点识别不同底物蛋白的结构基础。该发现将为Trx底物蛋白的预测提供新的重要思路。
(Ⅰ) Many xenobiotics have cytotoxic and/or genotoxic properties, and can damage cells in various ways. To eliminate toxic xenobiotics, living organisms have evolved a series of sophisticated detoxification strategies. In general, the detoxification process can be described as a three-phase reaction. In phase I, xenobiotics are activated by introduction of reactive functional groups. In phase II, they are neutralized by conjugation to chemical constituents through the reactive groups. In phase III, the conjugated xenobiotics are pumped out from cells after being metabolized via downstream pathways and eventually eliminated.
The glutathione S-transferases (GSTs) (EC 2.5.1.18) play a key role in phase II of enzymatic detoxification. These enzymes catalyze the reaction of xenobiotics with the thiolate group of glutathione (GSH), thereby neutralizing their electrophilic sites and raising the water-solubility of the products. Eukaryotes usually contain multiple GST paralogs with different catalytic activities, and a wide range of cellular functions. In mammals, cytosolic GSTs are dimeric proteins that are grouped into eight main-strain classes, primarily by sequence alignment: Alpha, Kappa, Pi, Mu, Theta, Zeta, Sigma and Omega. In addition, studies in non-mammalian species have revealed several new classes such as theΒeta class of bacteria, the Phi and Tau classes in plants, and the Delta class in insects. Fungi also possess several GSTs, but most cannot be grouped into the pre-existing classes. In addition to the sequence-based classification, the nomenclature of cytosolic GSTs can refer to the essential catalytic residue. All cytosolic GSTs of known structure are classified into one of three catalytic types, either Tyr-, Ser- or Cys-type, depending on the residue that drives the conjugation of GSH to a xenobiotic through lowering the pKa of GSH, and stabilizing the thiolate anion via a hydrogen bond. Mutations of the essential residue usually results in a substantial, if not complete, inactivation of the GST.
In the yeast Saccharomyces cerevisiae, seven proteins possess GST activity (Gtt1 and 2, Gto1, 2 and 3 and Grx1, 2). However, the structures of Grx1 and Grx2 reveal that they are not bona fide GSTs, but only correspond to the GST N-terminal domain. Of the five other homologous proteins, Gto1, 2 and 3 are classified into the Omega GST class, while Gtt1 and 2 are not categorized into any existing classes. Although the physiological and biochemical functions of these five yeast GSTs have been extensively studied, no three-dimensional (3-D) structures have been reported. We began a systematic characterization of the yeast GSTs by determining the crystal structures of Gtt2 in apo form at 2.23 (A|°), and in two ligand-bound forms at 2.20 and 2.10 (A|°). Gtt2 is distinct from the three classic catalytic types, because a water molecule fixed by two residues in the C-terminal domain is responsible for the activity. Moreover, only glycine and alanine are favored at the N-terminus of helixα1 because of the stereo-hindrance. These results enabled us to define a novel catalytic cytosolic GST type, which is called the atypical-type.
(Ⅱ) Thioredoxins (Trxs) are ubiquitous small thiol-disulfide exchange proteins which are involved in many important cellular processes such as reduction of methionine sulfoxide, ribonucleotide, and peroxide. These proteins have a highly conserved active site of CXXC motif. During reaction, the first Cys attacks the intramolecular disulfide bond in the substrate protein, accompanying with the formation of an intermolecular disulfide intermediate. This mixed disulfide is subsequently attacked by the second Cys, resulting in the release of reduced substrate protein and the oxidized Trx.
Methionine is one of the most sensitive amino acid residues subject to oxidation. It can be readily oxidized to methionine sulfoxide (Met-SO) as a mixture of two enantiomers at the sulfoxide moiety (Met-S-SO and Met-R-SO), by various reactive oxygen or nitrogen species. Oxidation of the methionines on protein surfaces would cause some lethal effects to the cells and accelerate the aging process. However, both in vitro and in vivo, a group of enzymes called methionine sulfoxide reductases (Msrs, EC 1.8.4.6) can use thioredoxin (Trx) as the electron donor to regenerate the oxidized proteins, by reducing Met-SO to methionine. Recently, the glutathione/glutaredoxin system has been discovered that it also could act as the electron donor for Met-SO reduction. This protective mechanism has been shown to play a significant role in elongating the lifespan of yeast, insects and mammals.
To date, three Msr families have been reported. MsrA and MsrB are classic Msrs that regenerate the proteinous Met-S-SO and Met-R-SO, respectively. They also could regenerate the corresponding free Met-SO. A series of human disease-related proteins have been identified as substrates of MsrA and MsrB, such as calmodulin, HIV-2 protease and alpha-1-proteinase inhibitor. However, fRMsr is a recently discovered Msr which is exclusively responsible for the reduction of free Met-R-SO, but not the proteinous ones. Despite that the three families have distinct differences in origin, structure, substrate specificity and species distribution, they basically share a similar catalytic mechanism. The mechanism involves the oxidation of the catalytic cysteine to a sulfenic acid intermediate, followed by the formation of an intramolecular disulfide bond, and the final regeneration process driven by Trx or other reductants.
Structures of MsrA from seven different species are currently reported. The core structures of these MsrA are very similar and could be well superimposed. This is regardless of the method of structure determination, either crystallography or NMR spectroscopy. The major differences are involved in the loops around the active site, especially the C-terminal loop, which is supported by the electrostatic analysis and distance measurements between the two catalytic cysteines. It is also suggested that these conformational changes would facilitate the formation of an intramolecular disulfide bond and the exposure of a hydrophobic patch for Trx interaction.
The yeast Saccharomyces cerevisiae encodes three Msrs (Mxr1/MsrA, Mxr2/MsrB and Ykg9/fRMsr). Although the mechanism of the Msrs has been extensively studied, their interactions with diverse substrate proteins and a universal Trx remain unknown. Therefore, we have systematically characterized the yeast Msr-Trx complexes by determining the crystal structures of Mxr1 in its reduced form at 2.04 (A|°), in a dimeric oxidized form at 1.90 (A|°), and in a Trx2-complexed form at 2.70 (A|°). Superposition of these structures have revealed three highly flexible loops. These loops undergo drastic conformational changes and may be responsible for the enzyme’s substrate diversity. Additionally, interface analysis of Mxr1-Trx2 along with data from previously reported Trx-protein complexes, we have found a new mode for Trx-involved complexes which undergo drastic conformational changes. These findings would be helpful for the prediction of potential interfaces on the Trx substrate proteins.
谷胱甘肽转移酶(glutathione S-transferase,GST,EC 2.5.1.18)就是在解毒第二阶段中发挥重要作用的一种酶。该家族的酶能够催化异生质与谷胱甘肽(glutathione,GSH)巯基的偶联,进而消除异生质的亲电基团,降低毒性,提高溶解度。真核生物通常含有多个直系同源的谷胱甘肽转移酶,参与不同的反应流程。在哺乳动物中,胞质内的谷胱甘肽转移酶全都是以二体形式存在,并根据一级序列被划分为八个主要亚家族:Alpha、Kappa、Pi、Mu、Theta、Zeta、Sigma和Omega。在对非哺乳动物的研究中,一些新的谷胱甘肽转移酶亚家族被报道,如细菌内的Beta亚家族,高等植物中的Phi和Tau亚家族,昆虫中的Delta亚家族。真菌也含有多种谷胱甘肽转移酶,但是一级序列变化较大,目前无法归入依照序列划分的分类体系。除了这种分类体系,还有一种根据谷胱甘肽转移酶催化残基的分类方法。谷胱甘肽转移酶在催化过程中,将依靠某一特定催化残基去降低谷胱甘肽的pKa值,通过氢键稳定巯基负离子,从而加速异生质的亲电攻击。该分类方法依照反应过程中催化残基的类型,将所有的胞内谷胱甘肽转移酶分为三大类:酪氨酸催化(Tyr-),丝氨酸催化(Ser-),半胱氨酸催化(Cys-)。目前所有的胞内谷胱甘肽转移酶都可以归入上述三个大类。据报道,催化残基的突变可以显著降低,甚至完全消除谷胱甘肽转移酶的活性。
在酿酒酵母(Saccharomyces cerevisiae)中,目前发现七种蛋白具有谷胱甘肽转移酶活性,分别是Gtt1、Gtt2、Gto1、Gto2、Gto3、Grx1和Grx2。从已知的结构看,Grx1和Grx2并不属于谷胱甘肽转移酶家族,因为它们没有谷胱甘肽转移酶的C端结构域,而只具有N端结构域。在剩下的五个蛋白均属于谷胱甘肽转移酶家族,其中Gto1/2/3被归入谷胱甘肽转移酶Omega亚家族,Gtt1和Gtt2因序列同源度过低而无法归入现有的亚家族。虽然这五个谷胱甘肽转移酶的生理和生化功能都被广泛研究,但是到目前为止它们的三维结构还没有被报道。因此,我们展开了一项关于酿酒酵母谷胱甘肽转移酶三维结构的研究。我们解析了酿酒酵母中谷胱甘肽转移酶Gtt2的结构(2.23 (A|°)),以及Gtt2分别与底物谷胱甘肽GSH(2.10 (A|°))和底物类似物磺化谷胱甘肽GTS(2.20 (A|°))的复合物结构。从整体结构上看,Gtt2与常见的谷胱甘肽转移酶没有太大区别。但是通过对催化位点的结构分析、定点突变、光谱学分析和酶活测定,我们发现Gtt2与前人报道的三种催化类型(Tyr-、Ser-、Cys-)显著不同。Gtt2是通过C端结构域上的一个丝氨酸和一个组氨酸共同稳定的水分子来发挥催化功能的,而不是通过某个催化残基上的极性原子。另外,同源序列比对、定点突变与酶活测定证明,伸向催化位点的α1螺旋的N端,由于空间位阻的原因,只能是甘氨酸或者丙氨酸这两种侧链较小的残基。最终,我们定义了一类新的谷胱甘肽转移酶催化类型:非典型性(Atypical-type)谷胱甘肽转移酶。
(Ⅱ)硫氧还蛋白(thioredoxin,Trx)是一类广泛存在于自然界中的“巯基—二硫键”交换蛋白。它参与了很多重要的胞内反应,比如还原甲硫氨酸亚砜、核糖核苷酸、过氧化物等等。硫氧还蛋白家族都具有一个高度保守的CXXC活性相关结构域。反应过程中,第一个半胱氨酸会攻击底物蛋白的分子内二硫键,将分子内二硫键转化成分子间二硫键,形成反应中间态。此时硫氧还蛋白和底物蛋白会形成一个极不稳定的二硫键连接的复合物。之后,该分子间二硫键会被硫氧还蛋白CXXC中的第二个Cys攻击,中间态复合物解体,释放出被还原了的底物蛋白和被氧化了的硫氧还蛋白。
甲硫氨酸是一种对氧化非常敏感的氨基酸。在活性氧簇或活性氮簇存在时,甲硫氨酸很容易被氧化成甲硫氨酸亚砜(Met-SO)。由于是手性分子,所以氧化产物是以镜像对映体的形式(Met-S-SO and Met-R-SO)混合存在。蛋白表面的甲硫氨酸被氧化后会对细胞造成一系列损伤并最终加速衰老过程。然而,细胞内有一种称为甲硫氨酸亚砜还原酶(methionine-S-sulfoxide reductase,Msr)的蛋白,能够用硫氧还蛋白Trx提供的电子,去还原被氧化生成的甲硫氨酸亚砜,重新变成甲硫氨酸。最近,法国一科研小组报道,不光是硫氧还蛋白可以给甲硫氨酸亚砜还原酶提供电子,谷氧还蛋白/谷胱甘肽系统同样可以作为还原力的提供者。不论其还原力来源于哪里,甲硫氨酸亚砜还原酶都被证明在酿酒酵母、昆虫、哺乳动物体内,有延长寿命的作用。
到目前为止,一共有三个甲硫氨酸亚砜还原酶家族被报道(MsrA、MsrB、fRMsr)。MsrA和MsrB是最先被发现的两个家族,对底物有手性要求,分别能还原Met-S-SO和Met-R-SO。他们不但能够还原游离状态的甲硫氨酸亚砜,还能够还原蛋白表面的甲硫氨酸亚砜。据报道,一系列与人类疾病相关的蛋白都被证明是MsrA和MsrB的底物,比如钙调蛋白、HIV-2蛋白酶、Alpha-1蛋白酶抑制剂等等。fRMsr家族直到最近才被发现,它的特点是只能够还原游离状态的Met-R-SO,对蛋白表面的Met-R-SO则无能为力。尽管这三个家族有不同的起源、结构、底物特异性和物种分布,但它们所涉及的催化机理却是基本一致的。都是甲硫氨酸亚砜被还原并释放,催化半胱氨酸的巯基氧化成次磺酸,转化为分子内二硫键,最后由硫氧还蛋白或者其他电子供体还原Msr的分子内二硫键。
现在已经有七个不同物种的MsrA结构被报道。不论是X射线晶体衍射方法解析的还是核磁共振波谱法解析的,它们的核心结构域基本一致,可以很好的叠合。各结构之间的区别主要集中在活性位点周围的柔性区,尤其是C末端的柔性区,这恰恰是参与催化的一个半胱氨酸所在的位置,暗示这些柔性区的构象变化很可能会促进分子内二硫键的形成,并与硫氧还蛋白相互作用有关。
酿酒酵母(Saccharomyces cerevisiae)中存在三个甲硫氨酸亚砜还原酶,分别是Mxr1/MsrA、Mxr2/MsrB和Ykg9/fRMsr。尽管这三个甲硫氨酸亚砜还原酶的机制已经被广泛研究,但是它们为什么能够与多种的底物蛋白或者硫氧还蛋白相互作用,仍然还不清楚。为此,我们解析了Mxr1还原态(2.04 (A|°)),二体氧化态(1.90 (A|°))以及与硫氧还蛋白Trx2结合的复合物(2.70 (A|°))的晶体结构。通过结构叠合分析,我们发现活性位点周围的三段柔性区构象变化很大,可以根据底物蛋白的表面而改变构象,这很有可能就是甲硫氨酸亚砜还原酶底物多样性的原因。另外,该区域与Trx2相互作用时所显示的柔性,提供了Trx能用单一活性位点识别不同底物蛋白的结构基础。该发现将为Trx底物蛋白的预测提供新的重要思路。
(Ⅰ) Many xenobiotics have cytotoxic and/or genotoxic properties, and can damage cells in various ways. To eliminate toxic xenobiotics, living organisms have evolved a series of sophisticated detoxification strategies. In general, the detoxification process can be described as a three-phase reaction. In phase I, xenobiotics are activated by introduction of reactive functional groups. In phase II, they are neutralized by conjugation to chemical constituents through the reactive groups. In phase III, the conjugated xenobiotics are pumped out from cells after being metabolized via downstream pathways and eventually eliminated.
The glutathione S-transferases (GSTs) (EC 2.5.1.18) play a key role in phase II of enzymatic detoxification. These enzymes catalyze the reaction of xenobiotics with the thiolate group of glutathione (GSH), thereby neutralizing their electrophilic sites and raising the water-solubility of the products. Eukaryotes usually contain multiple GST paralogs with different catalytic activities, and a wide range of cellular functions. In mammals, cytosolic GSTs are dimeric proteins that are grouped into eight main-strain classes, primarily by sequence alignment: Alpha, Kappa, Pi, Mu, Theta, Zeta, Sigma and Omega. In addition, studies in non-mammalian species have revealed several new classes such as theΒeta class of bacteria, the Phi and Tau classes in plants, and the Delta class in insects. Fungi also possess several GSTs, but most cannot be grouped into the pre-existing classes. In addition to the sequence-based classification, the nomenclature of cytosolic GSTs can refer to the essential catalytic residue. All cytosolic GSTs of known structure are classified into one of three catalytic types, either Tyr-, Ser- or Cys-type, depending on the residue that drives the conjugation of GSH to a xenobiotic through lowering the pKa of GSH, and stabilizing the thiolate anion via a hydrogen bond. Mutations of the essential residue usually results in a substantial, if not complete, inactivation of the GST.
In the yeast Saccharomyces cerevisiae, seven proteins possess GST activity (Gtt1 and 2, Gto1, 2 and 3 and Grx1, 2). However, the structures of Grx1 and Grx2 reveal that they are not bona fide GSTs, but only correspond to the GST N-terminal domain. Of the five other homologous proteins, Gto1, 2 and 3 are classified into the Omega GST class, while Gtt1 and 2 are not categorized into any existing classes. Although the physiological and biochemical functions of these five yeast GSTs have been extensively studied, no three-dimensional (3-D) structures have been reported. We began a systematic characterization of the yeast GSTs by determining the crystal structures of Gtt2 in apo form at 2.23 (A|°), and in two ligand-bound forms at 2.20 and 2.10 (A|°). Gtt2 is distinct from the three classic catalytic types, because a water molecule fixed by two residues in the C-terminal domain is responsible for the activity. Moreover, only glycine and alanine are favored at the N-terminus of helixα1 because of the stereo-hindrance. These results enabled us to define a novel catalytic cytosolic GST type, which is called the atypical-type.
(Ⅱ) Thioredoxins (Trxs) are ubiquitous small thiol-disulfide exchange proteins which are involved in many important cellular processes such as reduction of methionine sulfoxide, ribonucleotide, and peroxide. These proteins have a highly conserved active site of CXXC motif. During reaction, the first Cys attacks the intramolecular disulfide bond in the substrate protein, accompanying with the formation of an intermolecular disulfide intermediate. This mixed disulfide is subsequently attacked by the second Cys, resulting in the release of reduced substrate protein and the oxidized Trx.
Methionine is one of the most sensitive amino acid residues subject to oxidation. It can be readily oxidized to methionine sulfoxide (Met-SO) as a mixture of two enantiomers at the sulfoxide moiety (Met-S-SO and Met-R-SO), by various reactive oxygen or nitrogen species. Oxidation of the methionines on protein surfaces would cause some lethal effects to the cells and accelerate the aging process. However, both in vitro and in vivo, a group of enzymes called methionine sulfoxide reductases (Msrs, EC 1.8.4.6) can use thioredoxin (Trx) as the electron donor to regenerate the oxidized proteins, by reducing Met-SO to methionine. Recently, the glutathione/glutaredoxin system has been discovered that it also could act as the electron donor for Met-SO reduction. This protective mechanism has been shown to play a significant role in elongating the lifespan of yeast, insects and mammals.
To date, three Msr families have been reported. MsrA and MsrB are classic Msrs that regenerate the proteinous Met-S-SO and Met-R-SO, respectively. They also could regenerate the corresponding free Met-SO. A series of human disease-related proteins have been identified as substrates of MsrA and MsrB, such as calmodulin, HIV-2 protease and alpha-1-proteinase inhibitor. However, fRMsr is a recently discovered Msr which is exclusively responsible for the reduction of free Met-R-SO, but not the proteinous ones. Despite that the three families have distinct differences in origin, structure, substrate specificity and species distribution, they basically share a similar catalytic mechanism. The mechanism involves the oxidation of the catalytic cysteine to a sulfenic acid intermediate, followed by the formation of an intramolecular disulfide bond, and the final regeneration process driven by Trx or other reductants.
Structures of MsrA from seven different species are currently reported. The core structures of these MsrA are very similar and could be well superimposed. This is regardless of the method of structure determination, either crystallography or NMR spectroscopy. The major differences are involved in the loops around the active site, especially the C-terminal loop, which is supported by the electrostatic analysis and distance measurements between the two catalytic cysteines. It is also suggested that these conformational changes would facilitate the formation of an intramolecular disulfide bond and the exposure of a hydrophobic patch for Trx interaction.
The yeast Saccharomyces cerevisiae encodes three Msrs (Mxr1/MsrA, Mxr2/MsrB and Ykg9/fRMsr). Although the mechanism of the Msrs has been extensively studied, their interactions with diverse substrate proteins and a universal Trx remain unknown. Therefore, we have systematically characterized the yeast Msr-Trx complexes by determining the crystal structures of Mxr1 in its reduced form at 2.04 (A|°), in a dimeric oxidized form at 1.90 (A|°), and in a Trx2-complexed form at 2.70 (A|°). Superposition of these structures have revealed three highly flexible loops. These loops undergo drastic conformational changes and may be responsible for the enzyme’s substrate diversity. Additionally, interface analysis of Mxr1-Trx2 along with data from previously reported Trx-protein complexes, we have found a new mode for Trx-involved complexes which undergo drastic conformational changes. These findings would be helpful for the prediction of potential interfaces on the Trx substrate proteins.
引文
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