在半乳糖氧化酶中通過銅介導的碳-氟鍵斷裂形成單氟取代的自由基輔因子
作者：
小柯機器人發布時間：2020/10/28 12:53:19
近日，德克薩斯大學聖安東尼奧分校的劉愛民課題組發現，在半乳糖氧化酶中，銅介導的碳氟鍵斷裂可以形成單氟取代的自由基輔因子。相關論文發表在2020年10月22日出版的《美國化學會志》上。
在該研究中，課題組人員通過擴展遺傳密碼的方法將3,5-二氯酪氨酸（Cl2-Tyr）和3,5-二氟酪氨酸（F2-Tyr）摻入通過定向進化優化表達的半乳糖氧化酶（GAOV）中，以取代原有的272號酪氨酸（Tyr272）。擁有這兩種非天然胺基酸的半乳糖氧化酶保留了催化活性。研究人員解析了GAOV、Cl2-Tyr272和F2-Tyr取代的GAOV的高解析度晶體結構，其解析度分別為1.48 Å、1.23 Å和1.80 Å。
結構數據顯示，在輔因子中僅保留了一個滷素取代原子，這表明在輔因子生物發生的自催化過程中，發生了碳-氯/氟鍵的氧化斷裂。用自由基清除劑羥基脲處理後，原本因自旋耦合隱藏的二價銅得以被電子自旋共振譜觀察到。因此，和野生型一樣，非天然酪氨酸取代後的酶結構上的催化中心仍包含自由基，即Cu(II)-(Cl-Tyr•-Cys)和Cu(II)-(F-Tyr•-Cys)。這些發現揭示了一種在生物中從未觀察到過的、由單核銅中心參與的碳-氯/碳-氟鍵斷裂。
據介紹，半乳糖氧化酶（GAO）包含一個二價銅-配體自由基輔因子。該輔因子中一個半胱氨酸-酪氨酸自由基與銅配位，通過銅的氧化以自催化的形式產生。交聯硫醚鍵的形成伴隨著Tyr272上的一個碳-氫鍵的斷裂，但目前對後者的具體細節知之甚少。
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