近日，德克萨斯大学圣安东尼奥分校的刘爱民课题组发现，在半乳糖氧化酶中，铜介导的碳氟键断裂可以形成单氟取代的自由基辅因子。相关论文发表在2020年10月22日出版的《美国化学会志》上。

在该研究中，课题组人员通过扩展遗传密码的方法将3,5-二氯酪氨酸（Cl₂-Tyr）和3,5-二氟酪氨酸（F₂-Tyr）掺入通过定向进化优化表达的半乳糖氧化酶（GAO^V）中，以取代原有的272号酪氨酸（Tyr272）。拥有这两种非天然氨基酸的半乳糖氧化酶保留了催化活性。研究人员解析了GAO^V、Cl₂-Tyr272和F₂-Tyr取代的GAO^V的高分辨率晶体结构，其分辨率分别为1.48 Å、1.23 Å和1.80 Å。

结构数据显示，在辅因子中仅保留了一个卤素取代原子，这表明在辅因子生物发生的自催化过程中，发生了碳-氯/氟键的氧化断裂。用自由基清除剂羟基脲处理后，原本因自旋耦合隐藏的二价铜得以被电子自旋共振谱观察到。因此，和野生型一样，非天然酪氨酸取代后的酶结构上的催化中心仍包含自由基，即Cu(II)-(Cl-Tyr^•-Cys)和Cu(II)-(F-Tyr^•-Cys)。这些发现揭示了一种在生物中从未观察到过的、由单核铜中心参与的碳-氯/碳-氟键断裂。

据介绍，半乳糖氧化酶（GAO）包含一个二价铜-配体自由基辅因子。该辅因子中一个半胱氨酸-酪氨酸自由基与铜配位，通过铜的氧化以自催化的形式产生。交联硫醚键的形成伴随着Tyr272上的一个碳-氢键的断裂，但目前对后者的具体细节知之甚少。

附：英文原文

Title: Formation of Monofluorinated Radical Cofactor in Galactose Oxidase through Copper-Mediated C–F Bond Scission

Author: Jiasong Li, Ian Davis, Wendell P. Griffith, Aimin Liu

Issue&Volume: October 22, 2020

Abstract: Galactose oxidase (GAO) contains a Cu(II)-ligand radical cofactor. The cofactor, which is autocatalytically generated through the oxidation of the copper, consists of a cysteine-tyrosine radical (Cys-Tyr) as a copper ligand. The formation of the cross-linked thioether bond is accompanied by a C–H bond scission on Tyr272 with few details known thus far. Here, we report the genetic incorporation of 3,5-dichlorotyrosine (Cl2-Tyr) and 3,5-difluorotyrosine (F2-Tyr) to replace Tyr272 in the GAOV previously optimized for expression through directed evolution. The proteins with an unnatural tyrosine residue are catalytically competent. We determined the high-resolution crystal structures of the GAOV, Cl2-Tyr272, and F2-Tyr272 incorporated variants at 1.48, 1.23, and 1.80 resolution, respectively. The structural data showed only one halogen remained in the cofactor, indicating that an oxidative carbon-chlorine/fluorine bond scission has occurred during the autocatalytic process of cofactor biogenesis. Using hydroxyurea as a radical scavenger, the spin-coupled hidden Cu(II) was observed by EPR spectroscopy. Thus, the structurally defined catalytic center with genetic unnatural tyrosine substitution is in the radical containing form as in the wild-type, i.e., Cu(II)-(Cl-Tyr-Cys) or Cu(II)-(F-Tyr-Cys). These findings illustrate a previously unobserved C–F/C–Cl bond cleavage in biology mediated by a mononuclear copper center.

DOI: 10.1021/jacs.0c08992

Source: https://pubs.acs.org/doi/10.1021/jacs.0c08992