Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.
Department of Chemical Engineering and Analytical Science, The University of Manchester, Oxford Road, Manchester M13 9PL, United Kingdom.
Inorg Chem. 2021 Apr 5;60(7):4800-4815. doi: 10.1021/acs.inorgchem.0c03749. Epub 2021 Mar 25.
The carbon starvation-induced protein D (CsiD) is a recently characterized iron(II)/α-ketoglutarate-dependent oxygenase that activates a glutarate molecule as substrate at the C position to exclusively produce ()-2-hydroxyglutarate products. This selective hydroxylation reaction by CsiD is an important component of the lysine biodegradation pathway in ; however, little is known on the details and the origin of the selectivity of the reaction. So far, experimental studies failed to trap and characterize any short-lived catalytic cycle intermediates. As no computational studies have been reported on this enzyme either, we decided to investigate the chemical reaction mechanism of glutarate activation by an iron(IV)-oxo model of the CsiD enzyme. In this work, we present a density functional theory study on a large active site cluster model of CsiD and investigate the glutarate hydroxylation pathways by a high-valent iron(IV)-oxo species leading to ()-2-hydroxyglutarate, ()-2-hydroxyglutarate, and 3-hydroxyglutarate. In agreement with experimental observation, the favorable product channel leads to pro- C-H hydrogen atom abstraction to form ()-2-hydroxyglutarate. The reaction is stepwise with a hydrogen atom abstraction by an iron(IV)-oxo species followed by OH rebound from a radical intermediate. The work presented in this paper shows that despite the fact that the C-H bond strengths at the C and C positions of glutarate are similar in the gas phase, substrate binding and positioning guide the reaction to an enantioselective reaction process by destabilizing the hydrogen atom abstraction pathways for the pro- C-H and C-H positions. Our studies predict the chemical properties of the iron(IV)-oxo species and its rate constants with glutarate and deuterated-glutarate. Moreover, the work shows little protein motions during the catalytic process, while the substrate entrance into the substrate binding pocket appears to be guided by three active site arginine residues that position the substrate for pro- C-H hydrogen atom abstraction. Finally, the calculations show that irrespective of the position of the substrate and what C-H bond is closest to the metal center, the lowest energy pathway is for a selective pro- C-H hydrogen atom abstraction.
碳饥饿诱导蛋白 D (CsiD) 是一种最近被描述的铁(II)/α-酮戊二酸依赖性加氧酶，它能激活谷氨酸分子作为 C 位的底物，专门生成 ()-2-羟基戊二酸产物。CsiD 的这种选择性羟化反应是赖氨酸生物降解途径中的一个重要组成部分；然而，关于反应的细节和选择性的来源知之甚少。到目前为止，实验研究未能捕获和表征任何短寿命的催化循环中间体。由于也没有关于该酶的计算研究，我们决定研究 CsiD 酶的铁(IV)-氧模型对谷氨酸激活的化学反应机制。在这项工作中，我们对 CsiD 的一个大活性位点簇模型进行了密度泛函理论研究，并通过高氧化态铁(IV)-氧物种研究了导致 ()-2-羟基戊二酸、()-2-羟基戊二酸和 3-羟基戊二酸的谷氨酸羟化途径。与实验观察一致，有利的产物通道导致前 C-H 氢原子的提取，形成 ()-2-羟基戊二酸。该反应是分步进行的，铁(IV)-氧物种首先进行氢原子提取，然后从自由基中间体中进行 OH 回跳。本文介绍的工作表明，尽管在气相中谷氨酸的 C 和 C 位置的 C-H 键强度相似，但底物结合和定位通过使前 C-H 和 C-H 位置的氢原子提取途径失稳，引导反应进行对映选择性反应过程。我们的研究预测了铁(IV)-氧物种及其与谷氨酸和氘代谷氨酸的反应速率常数的化学性质。此外，该工作表明在催化过程中几乎没有蛋白质运动，而底物进入底物结合口袋似乎由三个活性位点精氨酸残基引导，这些残基将底物定位在前 C-H 氢原子提取位置。最后，计算表明，无论底物的位置和最接近金属中心的 C-H 键是什么，最低能量途径都是选择性的前 C-H 氢原子提取。
