Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Proc Natl Acad Sci U S A. 2013 Dec 17;110(51):20485-90. doi: 10.1073/pnas.1313620110. Epub 2013 Dec 2.
Hydrogenase pleiotropically acting protein (Hyp)E plays a role in biosynthesis of the cyano groups for the NiFe(CN)2CO center of [NiFe] hydrogenases by catalyzing the ATP-dependent dehydration of the carbamoylated C-terminal cysteine of HypE to thiocyanate. Although structures of HypE proteins have been determined, until now there has been no structural evidence to explain how HypE dehydrates thiocarboxamide into thiocyanate. Here, we report the crystal structures of the carbamoylated and cyanated forms of HypE from Thermococcus kodakarensis in complex with nucleotides at 1.53- and 1.64-Å resolution, respectively. Carbamoylation of the C-terminal cysteine (Cys338) of HypE by chemical modification is clearly observed in the present structures. In the presence of ATP, the thiocarboxamide of Cys338 is successfully dehydrated into the thiocyanate. In the carbamoylated state, the thiocarboxamide nitrogen atom of Cys338 is close to a conserved glutamate residue (Glu272), but the spatial position of Glu272 is less favorable for proton abstraction. On the other hand, the thiocarboxamide oxygen atom of Cys338 interacts with a conserved lysine residue (Lys134) through a water molecule. The close contact of Lys134 with an arginine residue lowers the pKa of Lys134, suggesting that Lys134 functions as a proton acceptor. These observations suggest that the dehydration of thiocarboxamide into thiocyanate is catalyzed by a two-step deprotonation process, in which Lys134 and Glu272 function as the first and second bases, respectively.
氢酶多功能作用蛋白（Hyp）E 通过催化 HypE 的氨甲酰化 C 末端半胱氨酸的 ATP 依赖性脱水作用，在 [NiFe] 氢化酶的氰基基团的生物合成中发挥作用，生成氰基硫代甲酰胺。尽管已经确定了 HypE 蛋白的结构，但到目前为止，还没有结构证据可以解释 HypE 如何将硫代羧酰胺脱水成硫氰酸盐。在这里，我们报道了来自 Thermococcus kodakarensis 的 HypE 的氨甲酰化和氰化形式与核苷酸在 1.53-和 1.64-Å分辨率下的复合物的晶体结构。HypE 的 C 末端半胱氨酸（Cys338）的氨甲酰化通过化学修饰在目前的结构中清晰可见。在 ATP 的存在下，Cys338 的硫代羧酰胺成功地脱水生成硫氰酸盐。在氨甲酰化状态下，Cys338 的硫代羧酰胺氮原子靠近保守的谷氨酸残基（Glu272），但 Glu272 的空间位置不利于质子的提取。另一方面，Cys338 的硫代羧酰胺氧原子通过一个水分子与保守的赖氨酸残基（Lys134）相互作用。Lys134 与精氨酸残基的紧密接触降低了 Lys134 的 pKa，表明 Lys134 作为质子受体发挥作用。这些观察结果表明，硫代羧酰胺的脱水反应是由两步去质子化过程催化的，其中 Lys134 和 Glu272 分别作为第一和第二碱起作用。
