Bichet Andreas, Hannemann Frank, Rekowski Matthias, Bernhardt Rita
FR 8.3--Biochemie, Universität des Saarlandes, D-66041 Saarbrücken, Germany.
Protein Eng Des Sel. 2007 Mar;20(3):117-23. doi: 10.1093/protein/gzm002. Epub 2007 Feb 9.
Cytochromes P450 are involved in the biosynthesis of steroid hormones in mitochondria of the adrenal gland. The electrons required for these reactions are provided via a redox chain consisting of adrenodoxin reductase (AdR) and adrenodoxin (Adx). A prerequisite for a fast and efficient electron transfer as well as high catalytic activity is the formation of functional complexes between the different redox partners. To improve the protein-protein interactions by directed evolution, we developed a new in vivo selection system. This high-throughput screening method is based on the yeast two-hybrid system. It enables a background-free screening for increased protein-protein interactions between stable and functional species including cofactor-containing proteins (FAD, [2Fe-2S], heme). The method was successfully applied for the directed evolution of Adx and selected variants were analyzed biochemically and biophysically. All analyzed proteins exhibit typical characteristics of [2Fe-2S]-cluster-type ferredoxins. Adx-dependent substrate conversion assays with different cytochromes demonstrated that the improved ability of the mutants to form complexes results in an enhanced catalytic efficiency of the cytochrome P450 system.
细胞色素P450参与肾上腺线粒体中甾体激素的生物合成。这些反应所需的电子通过由肾上腺皮质铁氧还蛋白还原酶（AdR）和肾上腺皮质铁氧还蛋白（Adx）组成的氧化还原链提供。不同氧化还原伙伴之间形成功能复合物是快速高效电子转移以及高催化活性的前提条件。为了通过定向进化改善蛋白质-蛋白质相互作用，我们开发了一种新的体内筛选系统。这种高通量筛选方法基于酵母双杂交系统。它能够在无背景的情况下筛选稳定且有功能的物种（包括含辅因子的蛋白质（FAD、[2Fe-2S]、血红素））之间增强的蛋白质-蛋白质相互作用。该方法已成功应用于Adx的定向进化，并对筛选出的变体进行了生化和生物物理分析。所有分析的蛋白质都表现出[2Fe-2S]簇型铁氧还蛋白的典型特征。用不同细胞色素进行的依赖Adx的底物转化试验表明，突变体形成复合物能力的提高导致细胞色素P450系统的催化效率增强。
