Role of Secondary Coordination Sphere Residues in Halogenation Catalysis of Non-heme Iron Enzymes
Role of Secondary Coordination Sphere Residues in Halogenation Catalysis of Non-heme Iron Enzymes
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二级配位球残基在非血红素铁酶卤化催化中的作用
DOI：
10.1021/acscatal.2c00954
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发表时间：
2022
期刊：
影响因子：
12.9
通讯作者：
Bhagi-Damodaran, Ambika
中科院分区：
文献类型：
--
作者：
Wilson, R. Hunter;Chatterjee, Sourav;Smithwick, Elizabeth R.;Dalluge, Joseph J.;Bhagi-Damodaran, Ambika
C(SP3)-H卤代反应的化学和区域选择性催化是化学合成中一个艰巨的目标。依赖于2-羟基戊二酸(2OG)的非血红素铁卤化酶催化C-H键的选择性氯化/溴化，并显示出与非血红素铁羟基酶高度的序列和结构相似性。这两个酶系统的次级配位球(SCS)是如何区分和确定它们的反应活性的，目前还不清楚。在这项工作中，我们证明了氧化还原活性酪氨酸残基在非血红素卤化铁酶的SCS中的特定位置对其结构、功能和反应性有巨大的影响。我们发现，在SyrB2卤化酶中，一个酪氨酸残基(F121Y)合理地结合到铁的氯化配体的氢键上，在生理上经历了翻译后的氧化生成二羟基苯丙氨酸(DOPA)。光谱、质谱学和生化研究相结合的结果表明，SyrB2中的DOPA修饰导致该酶不起作用。生物信息学分析表明，与羟基酶不同，SyrB2类卤代酶在121位具有保守的苯丙氨酸位置，以阻止这种无效的氧化。此外，分子动力学模拟结合DOPA仅在121位掺入的实验证明，使我们能够唯一地确定在SyrB2中起作用的是轴-氯卤代铁基异构体。我们还在其他依赖于20G的非血红素铁卤化酶的SCS中发现了保守的氧化还原失活残基，以避免DOPA样的非生产性氧化。总之，本研究证明了SCS在控制非血红素铁卤化酶结构和酶活性方面的重要性，并将对小分子和基于蛋白质的卤化催化剂的设计具有重要意义。
Chemo- and regio-selective catalysis of the C(sp3)-H halogenation reaction is a formidable goal in chemical synthesis. 2-Oxoglutarate (2OG)-dependent non-heme iron halogenases catalyze selective chlorination/bromination of C–H bonds and exhibit high sequence and structural similarities with non-heme iron hydroxylases. How the secondary coordination sphere (SCS) of these two enzyme systems differentiate and determine their reactivity is not well understood. In this work, we show that specific positioning of redox-active tyrosine residues in the SCS of non-heme iron halogenases has a huge impact on their structure, function, and reactivity. We discover that a tyrosine residue (F121Y) rationally incorporated to hydrogen bond to iron’s chloride ligand in SyrB2 halogenase undergoes post-translational oxidation to dihydroxyphenylalanine (DOPA) physiologically. A combination of spectroscopic, mass-spectrometric, and biochemical studies demonstrate that DOPA modification in SyrB2 renders the enzyme non-functional. Bioinformatic analysis suggests that SyrB2-like halogenases, unlike hydroxylases, have a conserved placement of phenylalanine at position 121 to preclude such unproductive oxidation. Furthermore, molecular dynamics simulations in tandem with experimental demonstration of DOPA incorporation exclusively at position 121 enables us to uniquely identify that an axial-chloro haloferryl isomer is operant in SyrB2. We also identify conserved redox-inactive residues in the SCS of other 2OG-dependent non-heme iron halogenases to avoid DOPA-like unproductive oxidations. Overall, this study demonstrates the importance of the SCS in controlling the structure and enzymatic activity of non-heme iron halogenases and will have significant implications toward the design of small-molecule and protein-based halogenation catalysts.