Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States.
Biochemistry. 2011 Feb 15;50(6):1091-100. doi: 10.1021/bi101741v. Epub 2011 Jan 20.
High-resolution crystal structures are reported for apo, holo, and substrate-bound forms of a toxoflavin-degrading metalloenzyme (TflA). In addition, the degradation reaction is shown to be dependent on oxygen, Mn(II), and dithiothreitol in vitro. Despite its low sequence identity with proteins of known structure, TflA is structurally homologous to proteins of the vicinal oxygen chelate superfamily. Like other metalloenzymes in this superfamily, the TflA fold contains four modules that associate to form a metal binding site; however, the fold displays a rare rearrangement of the structural modules indicative of domain permutation. Moreover, unlike the 2-His-1-carboxylate facial triad commonly utilized by vicinal oxygen chelate dioxygenases and other dioxygen-activating non-heme Fe(II) enzymes, the metal center in TflA consists of a 1-His-2-carboxylate facial triad. The substrate-bound complex shows square-pyramidal geometry in which one position is occupied by O5 of toxoflavin. The open coordination site is predicted to be the dioxygen binding site. TflA appears to stabilize the reduced form of toxoflavin through second-sphere interactions. This anionic species is predicted to be the electron source responsible for reductive activation of oxygen to produce a peroxytoxoflavin intermediate.
高分辨率晶体结构报告了脱辅基、全酶和底物结合形式的噻嗪黄素降解金属酶（TflA）。此外，体外降解反应依赖于氧气、Mn(II)和二硫苏糖醇。尽管 TflA 与已知结构的蛋白质序列同一性较低，但它在结构上与邻氧螯合超家族的蛋白质具有同源性。与该超家族中的其他金属酶一样，TflA 折叠包含四个模块，这些模块结合形成一个金属结合位点；然而，该折叠显示出结构模块罕见的重排，表明结构域的排列发生了变化。此外，与邻氧螯合双加氧酶和其他双加氧激活非血红素 Fe(II)酶常用的 2-His-1-carboxylate 面三角不同，TflA 中的金属中心由 1-His-2-carboxylate 面三角组成。底物结合复合物显示出四方锥几何形状，其中一个位置被噻嗪黄素的 O5 占据。开放的配位位点预计是双氧的结合位点。TflA 似乎通过二级相互作用稳定还原型噻嗪黄素。这种阴离子物种预计是负责将氧气还原为过氧噻嗪黄素中间体的电子源。
