Thioredoxin A active-site mutants form mixed disulfide dimers that resemble enzyme-substrate reaction intermediates.
Thioredoxin A active-site mutants form mixed disulfide dimers that resemble enzyme-substrate reaction intermediates.
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DOI：
10.1016/j.jmb.2008.03.077
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发表时间：
2008-06-06
影响因子：
5.6
通讯作者：
van Dijl, Jan Maarten
中科院分区：
文献类型：
--
作者：
Kouwen, Thijs R. H. M.;Andrell, Jun;Schrijver, Rianne;Dubois, Jean-Yves F.;Maher, Megan J.;Iwata, So;Carpenter, Elisabeth P.;van Dijl, Jan Maarten
硫氧还蛋白在几乎所有生物体中作为细胞溶质内的主要硫醇-二硫化物氧化还原酶发挥作用。其主要目的是通过在二硫键交换反应中将分子内二硫键转化为二硫醇来维持含半胱氨酸的蛋白质处于还原状态。据报道，硫氧还蛋白通过与不同细胞类型中的特定底物相互作用而促进多种生理功能。为了研究低GC革兰氏阳性菌枯草芽孢杆菌中必需硫氧还蛋白A（TrxA）的功能，我们纯化了野生型TrxA和三种突变体TrxA蛋白，它们在CxxC活性位点中缺少两个半胱氨酸残基中的一个或两个。纯蛋白质用于底物结合研究，称为“混合二硫键捕捞”，其中共价二硫键键键合的反应中间体可以可视化。一个前所未有的发现是，两个活性位点的半胱氨酸残基可以形成混合二硫化物与底物蛋白质时，其他活性位点的半胱氨酸是不存在的，但只有N-末端的活性位点的半胱氨酸形成稳定的相互作用。第二个新奇是，由于剩余的活性位点半胱氨酸残基的巯基氧化，两个单半胱氨酸突变体TrxA蛋白形成稳定的同源二聚体。为了研究这些二聚体是否类似于混合的酶-底物二硫化物，最丰富的二聚体C32 S的结构，其特征在于通过X射线晶体学。这产生了来自低GC革兰氏阳性细菌的硫氧还蛋白同源二聚体的高分辨率（1.5 μ m）X射线晶体结构。C32 S TrxA二聚体可被视为硫氧还蛋白的混合二硫键反应中间体，这揭示了硫氧还蛋白/底物结合模式的多样性。
Thioredoxin functions in nearly all organisms as the major thiol–disulfide oxidoreductase within the cytosol. Its prime purpose is to maintain cysteine-containing proteins in the reduced state by converting intramolecular disulfide bonds into dithiols in a disulfide exchange reaction. Thioredoxin has been reported to contribute to a wide variety of physiological functions by interacting with specific sets of substrates in different cell types. To investigate the function of the essential thioredoxin A (TrxA) in the low-GC Gram-positive bacterium Bacillus subtilis, we purified wild-type TrxA and three mutant TrxA proteins that lack either one or both of the two cysteine residues in the CxxC active site. The pure proteins were used for substrate-binding studies known as “mixed disulfide fishing” in which covalent disulfide-bonded reaction intermediates can be visualized. An unprecedented finding is that both active-site cysteine residues can form mixed disulfides with substrate proteins when the other active-site cysteine is absent, but only the N-terminal active-site cysteine forms stable interactions. A second novelty is that both single-cysteine mutant TrxA proteins form stable homodimers due to thiol oxidation of the remaining active-site cysteine residue. To investigate whether these dimers resemble mixed enzyme–substrate disulfides, the structure of the most abundant dimer, C32S, was characterized by X-ray crystallography. This yielded a high-resolution (1.5Å) X-ray crystallographic structure of a thioredoxin homodimer from a low-GC Gram-positive bacterium. The C32S TrxA dimer can be regarded as a mixed disulfide reaction intermediate of thioredoxin, which reveals the diversity of thioredoxin/substrate-binding modes.
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DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
3.6
作者：
Hochgräfe, F;Mostertz, J;Hecker, M
通讯作者：
Hecker, M
影响因子：
2.9
作者：
DYSON, HJ;GIPPERT, GP;WRIGHT, PE
通讯作者：
WRIGHT, PE
影响因子：
14.9
作者：
Davis IW;Leaver-Fay A;Chen VB;Block JN;Kapral GJ;Wang X;Murray LW;Arendall WB 3rd;Snoeyink J;Richardson JS;Richardson DC
通讯作者：
Richardson DC
影响因子：
2.9
作者：
Dyson, HJ;Jeng, MF;Holmgren, A
通讯作者：
Holmgren, A