Mechanistic Investigation of Isonitrile Formation Catalyzed by the Nonheme Iron/α-KG-Dependent Decarboxylase (ScoE)
Mechanistic Investigation of Isonitrile Formation Catalyzed by the Nonheme Iron/α-KG-Dependent Decarboxylase (ScoE)
复制标题
非血红素铁/α-KG 依赖性脱羧酶 (ScoE) 催化异腈形成的机理研究
DOI：
10.1021/acscatal.9b05411
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发表时间：
2020-03-06
期刊：
影响因子：
12.9
通讯作者：
Liu, Yongjun
中科院分区：
文献类型：
--
作者：
Li, Hong;Liu, Yongjun
最近的结构和生化证据表明，蓝淡红链霉菌的ScoE是一种非血红素铁/α-KG依赖的脱羧酶，它催化异腈基团通过去饱和和脱羧形成。这一发现为异腈的形成提供了另一种机制。其他异腈脱氢酶，如IsnA、XnPvcA或AmbI 1/AmbI 2，通过引入额外的碳单元将R-CH（-NH 2）-CO2-转化为R-CH（-NE C）CO2-;然而，ScoE通过氧化脱羧催化R-NH-CH 2-CO2-转化为R-NC。为了探索ScoE的催化机理，在高分辨晶体结构的基础上，构建了酶-底物复合物模型，并进行了一系列的QM/MM计算。我们的研究结果表明，SCOE催化的反应包含两个解耦的部分，去饱和和脱羧。Fe-IV-氧代触发的去饱和包括两个连续的H-提取，这类似于由其他非血红素铁/a-KG依赖性去饱和酶催化的C-C单键去饱和。在第二阶段反应中，由H-提取产生的底物自由基的脱羧被计算为相当容易，而先前提出的涉及羟基化中间体的脱羧被计算为困难。重要的是，从底物到铁中心的电子转移是降低脱羧屏障的关键因素。因此，中心铁离子不仅负责H-提取，而且还充当脱羧的电子中继站。此外，这种电子转移被认为是耦合与质子转移，其中R310和相关的H-键合网络发挥关键作用。一般来说，第一个C-N去饱和是整个催化反应的限速步骤，在两个竞争途径中的总能垒为17.6或16.9 kcal/mol，与实验估计的自由能（17.9-18.1 kcal/mol）定性一致。这些结果可能为了解异腈的生物合成和非血红素铁/α-KG依赖性酶催化的氧化脱羧提供有用的信息。
Recent structural and biochemical evidence showed that ScoE from Streptomyces coeruleorubidus is a nonheme iron/alpha-KG-dependent decarboxylase, which catalyzes the formation of isonitrile group by desaturation and decarboxylation. This discovery offers an alternative mechanism for isonitrile formation. The other isonitrile synthases, such as IsnA, XnPvcA, or AmbI1/AmbI2, convert R-CH(-NH2)-CO2- to R-CH(-NE C)CO2- by introducing an additional carbon unit; however, ScoE catalyzes the conversion of R-NH-CH2-CO2- to R-N C through oxidative decarboxylation. To explore the catalytic mechanism of ScoE, on the basis of the high-resolution crystal structure, the enzyme-substrate complex models were constructed and a series of combined QM/MM calculations were performed. Our results reveal that the ScoE-catalyzed reaction contains two decoupled parts, desaturation and decarboxylation. The Fe-IV-oxo-triggered desaturation includes two consecutive H-abstractions, which are similar to the C-C single bond desaturation catalyzed by other nonheme iron/a-KG-dependent desaturases. In the second stage reaction, the decarboxylation of the substrate radical generated by H-abstraction was calculated to be quite easy, whereas the previously proposed decarboxylation that involves the hydroxylated intermediate was calculated to be difficult. Importantly, the electron transfer from the substrate to the iron center is the key factor for lowering the barrier of decarboxylation. Thus, the central iron ion is not only responsible for H-abstraction but also acts as an electron relay station for decarboxylation. In addition, this electron transfer was found to be coupled with a proton transfer, in which R310 and the associated H-bonding network play a critical role. In general, the first C-N desaturation is the rate-limiting step of the whole catalysis with an overall energy barrier of 17.6 or 16.9 kcal/mol in two competitive pathways, qualitatively agreeing with the estimated free energy (17.9-18.1 kcal/mol) from experiments. These results may provide useful information for understanding the biosynthesis of isonitrile and the oxidative decarboxylation catalyzed by nonheme iron/a-KG-dependent enzymes.