Aldag Caroline, Gromov Igor A, García-Rubio Inés, von Koenig Konstanze, Schlichting Ilme, Jaun Bernhard, Hilvert Donald
Laboratory of Organic Chemistry, ETH Zurich, CH-8093 Zurich, Switzerland.
Proc Natl Acad Sci U S A. 2009 Apr 7;106(14):5481-6. doi: 10.1073/pnas.0810503106. Epub 2009 Mar 17.
The unique monooxygenase activity of cytochrome P450cam has been attributed to coordination of a cysteine thiolate to the heme cofactor. To investigate this interaction, we replaced cysteine with the more electron-donating selenocysteine. Good yields of the selenoenzyme were obtained by bacterial expression of an engineered gene containing the requisite UGA codon for selenocysteine and a simplified yet functional selenocysteine insertion sequence (SECIS). The sulfur-to-selenium substitution subtly modulates the structural, electronic, and catalytic properties of the enzyme. Catalytic activity decreases only 2-fold, whereas substrate oxidation becomes partially uncoupled from electron transfer, implying a more complex role for the axial ligand than generally assumed.
细胞色素P450cam独特的单加氧酶活性归因于半胱氨酸硫醇盐与血红素辅因子的配位作用。为了研究这种相互作用，我们用供电子能力更强的硒代半胱氨酸取代了半胱氨酸。通过细菌表达含有硒代半胱氨酸所需的UGA密码子和简化但功能正常的硒代半胱氨酸插入序列（SECIS）的工程基因，获得了高产率的硒酶。硫到硒的取代微妙地调节了该酶的结构、电子和催化特性。催化活性仅降低2倍，而底物氧化与电子转移部分解偶联，这意味着轴向配体的作用比通常认为的更为复杂。
