Cellular maturation of an iron-type nitrile hydratase interrogated using EPR spectroscopy
Cellular maturation of an iron-type nitrile hydratase interrogated using EPR spectroscopy
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DOI：
10.1007/s00775-019-01720-y
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发表时间：
2019-10-01
影响因子：
3
通讯作者：
Bennett, Brian
中科院分区：
文献类型：
--
作者：
Lankathilaka, K. P. Wasantha;Stein, Natalia;Bennett, Brian
腈水合酶（Nitrile hydratase，NHase）是一种非血红素含铁酶，其在商品化学合成、药物中间体合成和腈-（溴苯腈）污染土地的复垦中具有应用。该酶的机制研究已经复杂的多个重叠的Fe（III）EPR信号的表达。最近的个别信号被分配到不同的化学物种的DFT计算的帮助下。本研究旨在探讨过表达NHase的细胞中EPR信号的来源和演变，优化NHase研究用均质样品的制备方法，并探讨E.大肠杆菌过表达的酶的“绿色”化学。结果表明，腈水合酶在体内随时间形成两组无活性的复合物。一种是由于与内源性羧酸的可逆络合，而第二种是由于必需的半胱氨酸次磺酸的不可逆失活氧化。结果表明，可以通过采用厌氧方案来改善制剂的均匀性。通过EPR鉴定完整细胞中NHase的产物复合物，证明了底物丙烯腈和乙腈被细胞吸收并通过NHase水合为相应酰胺的能力。抑制剂丁酸和丁硼酸也被E.大肠杆菌和形成复合物与腈水合酶在体内，表明必须小心与环境变量时，尝试微生物辅助合成和回收。[图片]
Nitrile hydratase (NHase) is a non-heme iron-containing enzyme that has applications in commodity chemical synthesis, pharmaceutical intermediate synthesis, and reclamation of nitrile-(bromoxynil) contaminated land. Mechanistic study of the enzyme has been complicated by the expression of multiple overlapping Fe(III) EPR signals. The individual signals were recently assigned to distinct chemical species with the assistance of DFT calculations. Here, the origins and evolution of the EPR signals from cells overexpressing the enzyme were investigated, with the aims of optimizing the preparation of homogeneous samples of NHase for study and investigating the application of E. coli overexpressing the enzyme for "green" chemistry. It was revealed that nitrile hydratase forms two sets of inactive complexes in vivo over time. One is due to reversible complexation with endogenous carboxylic acids, while the second is due to irreversibly inactivating oxidation of an essential cysteine sulfenic acid. It was shown that the homogeneity of preparations can be improved by employing an anaerobic protocol. The ability of the substrates acrylonitrile and acetonitrile to be taken up by cells and hydrated to the corresponding amides by NHase was demonstrated by EPR identification of the product complexes of NHase in intact cells. The inhibitors butyric acid and butane boronic acid were also taken up by E. coli and formed complexes with NHase in vivo, indicating that care must be taken with environmental variables when attempting microbially assisted synthesis and reclamation.[GRAPHICS]