PDF(Pubmed)
Abstract：
The 21st amino acid, selenocysteine (Sec), is synthesized on its dedicated transfer RNA (tRNASec). In bacteria, Sec is synthesized from Ser-tRNA[Ser]Sec by Selenocysteine Synthase (SelA), which is a pivotal enzyme in the biosynthesis of Sec. The structural characterization of bacterial SelA is of paramount importance to decipher its catalytic mechanism and its role in the regulation of the Sec-synthesis pathway. Here, we present a comprehensive single-particle cryo-electron microscopy (SPA cryoEM) structure of the bacterial SelA with an overall resolution of 2.69 Å. Using recombinant Escherichia coli SelA, we purified and prepared samples for single-particle cryoEM. The structural insights from SelA, combined with previous in vivo and in vitro knowledge, underscore the indispensable role of decamerization in SelA\'s function. Moreover, our structural analysis corroborates previous results that show that SelA adopts a pentamer of dimers configuration, and the active site architecture, substrate binding pocket, and key K295 catalytic residue are identified and described in detail. The differences in protein architecture and substrate coordination between the bacterial enzyme and its counterparts offer compelling structural evidence supporting the independent molecular evolution of the bacterial and archaea/eukarya Ser-Sec biosynthesis present in the natural world.
摘要：
第21个氨基酸,硒代半胱氨酸（Sec），在其专用转移RNA(tRNASec)上合成。在细菌中，Sec是通过硒代半胱氨酸合成酶（SelA）从Ser-tRNA[Ser]Sec合成的，它是Sec生物合成中的关键酶。细菌SelA的结构表征对于破译其催化机理及其在Sec合成途径调节中的作用至关重要。这里,我们提供了细菌SelA的全面单粒子低温电子显微镜（SPAcryoEM）结构，总分辨率为2.69。使用重组大肠杆菌SelA，我们纯化并制备了单颗粒冷冻EM样品。来自SelA的结构见解，结合以前的体内和体外知识，强调了在SelA的功能中，解数化不可或缺的作用。此外，我们的结构分析证实了先前的结果表明，SelA采用二聚体构型的五聚体，和活动站点架构，底物结合袋，和关键的K295催化残留物进行了识别和详细描述。细菌酶与其对应物之间的蛋白质结构和底物协调的差异提供了令人信服的结构证据，支持自然界中存在的细菌和古细菌/真核生物Ser-Sec生物合成的独立分子进化。
