Wang Lei, Li Sheng-Jian, Sidhu Ateesh, Zhu Li, Liang Yi, Freedman Robert B, Wang Chih-Chen
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China, the Graduate School of the Chinese Academy of Sciences, Beijing 100049, China, the Department of Biological Sciences, Warwick University, Coventry CV4 7AL, United Kingdom, and the State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China; National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China, the Graduate School of the Chinese Academy of Sciences, Beijing 100049, China, the Department of Biological Sciences, Warwick University, Coventry CV4 7AL, United Kingdom, and the State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China, the Graduate School of the Chinese Academy of Sciences, Beijing 100049, China, the Department of Biological Sciences, Warwick University, Coventry CV4 7AL, United Kingdom, and the State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
J Biol Chem. 2009 Jan 2;284(1):199-206. doi: 10.1074/jbc.M806645200. Epub 2008 Nov 11.
Protein-disulfide isomerase (PDI), a critical enzyme responsible for oxidative protein folding in the eukaryotic endoplasmic reticulum, is composed of four thioredoxin domains a, b, b', a', and a linker x between b' and a'. Ero1-Lalpha, an oxidase for human PDI (hPDI), has been determined to have one molecular flavin adenine dinucleotide (FAD) as its prosthetic group. Oxygen consumption assays with purified recombinant Ero1-Lalpha revealed that it utilizes oxygen as a terminal electron acceptor producing one disulfide bond and one molecule of hydrogen peroxide per dioxygen molecule consumed. Exogenous FAD is not required for recombinant Ero1-Lalpha activity. By monitoring the reactivation of denatured and reduced RNase A, we reconstituted the Ero1-Lalpha/hPDI oxidative folding system in vitro and determined the enzymatic activities of hPDI in this system. Mutagenesis studies suggested that the a' domain of hPDI is much more active than the a domain in Ero1-Lalpha-mediated oxidative folding. A domain swapping study revealed that one catalytic thioredoxin domain to the C-terminal of bb'x, whether a or a', is essential in Ero1-Lalpha-mediated oxidative folding. These data, combined with a pull-down assay and isothermal titration calorimetry measurements, enabled the minimal element for binding with Ero1-Lalpha to be mapped to the b'xa' fragment of hPDI.
蛋白质二硫键异构酶（PDI）是真核细胞内质网中负责氧化蛋白质折叠的关键酶，由四个硫氧还蛋白结构域a、b、b'、a'以及b'和a'之间的连接区x组成。人PDI（hPDI）的氧化酶Ero1-Lalpha已被确定以一分子黄素腺嘌呤二核苷酸（FAD）作为其辅基。对纯化的重组Ero1-Lalpha进行的耗氧测定表明，它利用氧气作为末端电子受体，每消耗一分子双原子氧产生一个二硫键和一分子过氧化氢。重组Ero1-Lalpha的活性不需要外源FAD。通过监测变性和还原的核糖核酸酶A的再活化，我们在体外重建了Ero1-Lalpha/hPDI氧化折叠系统，并测定了该系统中hPDI的酶活性。诱变研究表明，在Ero1-Lalpha介导的氧化折叠中，hPDI的a'结构域比a结构域活性高得多。结构域交换研究表明，在Ero1-Lalpha介导的氧化折叠中，位于bb'x C末端的一个催化硫氧还蛋白结构域，无论是a还是a'，都是必不可少的。这些数据，结合下拉分析和等温滴定量热法测量，使得与Ero1-Lalpha结合的最小元件能够定位到hPDI的b'xa'片段。
