The 3-His Metal Coordination Site Promotes the Coupling of Oxygen Activation to Cysteine Oxidation in Cysteine Dioxygenase
The 3-His Metal Coordination Site Promotes the Coupling of Oxygen Activation to Cysteine Oxidation in Cysteine Dioxygenase
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DOI：
10.1021/acs.biochem.9b01085
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发表时间：
2020-06-02
期刊：
影响因子：
2.9
通讯作者：
Ellis, Holly R.
中科院分区：
文献类型：
--
作者：
Forbes, Dianna L.;Meneely, Kathleen M.;Ellis, Holly R.
半胱氨酸双加氧酶（CDO）在结构上类似于使用3-His/1-Glu配位方案的cupin酶。然而，谷氨酸配体被半胱氨酸（Cys 93）残基取代，其在生理条件下与酪氨酸（Tyr 157）形成硫醚键。为了重建cupin超家族中更常见的3-His/1-Glu金属配体，产生了回复变体C93 E CDO。该变体提供了用于测试Cys 93的结构和功能意义以及CDO中的交联的框架。虽然用C93 E CDO观察到分子氧消耗，但它不与L-半胱氨酸氧化偶联。底物类似物（D-半胱氨酸，半胱胺和3-巯基丙酸酯）是不可行的C93 E CDO变体的底物，虽然它们显示出可变的配位铁中心。C93 E和交联和非交联的野生型CDO的结构通过X射线晶体学分别解析到1.91、2.49和2.30埃。C93 E CDO变体与交联CDO相比具有相似的总体结构特性;然而，铁由3-His/1-Glu几何结构配位，仅留下两个配位位点可用于双氧和双齿L-半胱氨酸结合。Tyr 157的羟基在非交联和C93 E CDO中发生位移，这种位移阻止了残基参与底物稳定。基于这些结果，半胱氨酸双加氧酶的金属中心与许多cupin酶的3-His/1-Glu几何构型的偏离对于有效的底物结合是必要的。在CDO中用Cys取代Glu允许铁上的第三配位位点用于双齿半胱氨酸和单齿氧结合。
Cysteine dioxygenase (CDO) structurally resembles cupin enzymes that use a 3-His/1-Glu coordination scheme. However, the glutamate ligand is substituted with a cysteine (Cys93) residue, which forms a thioether bond with tyrosine (Tyr157) under physiological conditions. The reversion variant, C93E CDO, was generated in order to reestablish the more common 3-His/1-Glu metal ligands of the cupin superfamily. This variant provides a framework for testing the structural and functional significance of Cys93 and the cross-link in CDO. Although dioxygen consumption was observed with C93E CDO, it was not coupled with L-cysteine oxidation. Substrate analogues (D-cysteine, cysteamine, and 3-mercaptopropionate) were not viable substrates for the C93E CDO variant, although they showed variable coordinations to the iron center. The structures of C93E and cross-linked and non-cross-linked wild-type CDO were solved by X-ray crystallography to 1.91, 2.49, and 2.30 angstrom, respectively. The C93E CDO variant had similar overall structural properties compared to cross-linked CDO; however, the iron was coordinated by a 3-His/1-Glu geometry, leaving only two coordination sites available for dioxygen and bidentate L-cysteine binding. The hydroxyl group of Tyr157 shifted in both non-cross-linked and C93E CDO, and this displacement prevented the residue from participating in substrate stabilization. Based on these results, the divergence of the metal center of cysteine dioxygenase from the 3-His/1-Glu geometry seen with many cupin enzymes was essential for effective substrate binding. The substitution of Glu with Cys in CDO allows for a third coordination site on the iron for bidentate cysteine and monodentate oxygen binding.