Recombinant expression and characterisation of the oxygen-sensitive 2-enoate reductase from Clostridium sporogenes.
Recombinant expression and characterisation of the oxygen-sensitive 2-enoate reductase from Clostridium sporogenes.
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DOI：
10.1099/mic.0.000568
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发表时间：
2018-03
期刊：
影响因子：
--
通讯作者：
Stephens G
中科院分区：
文献类型：
--
作者：
Mordaka PM;Hall SJ;Minton N;Stephens G
烯还原酶在化学中间体和生物活性产物的制备中引起了极大的关注。迄今为止，研究主要集中在老黄酶样蛋白上，因为它们易于处理，而来自梭菌的2-烯酸还原酶受到的关注要少得多，因为它们的氧敏感性和缺乏合适的表达系统。在生孢梭菌DSM 795中鉴定了假设的2-烯酸还原酶基因fldZ。所编码的蛋白质与梭菌FMN-和FAD-依赖性2-烯酸还原酶（包括提出参与蛋白水解梭菌中的氨基酸代谢的肉桂酸还原酶）具有高度同源性。该基因被克隆并在大肠杆菌中过表达。成功的表达依赖于使用严格的厌氧条件下生长和酶的制备，因为FldZ是氧敏感的。该酶还原芳香族烯酸，如肉桂酸或对香豆酸，但不还原短链不饱和脂肪酸。β，β-二取代硝基烯烃（E）-1-硝基-2-苯基丙烯被还原为对映体纯的（R）-1-硝基-2-苯基丙烷，产率90%。 相比之下，α，β-二取代的硝基烯烃（E）-2-硝基-1-苯基丙烯以56%的中等产率和差的对映选择性（（S）-2-硝基-1-苯基丙烷为16%ee）被还原。 这种重组梭菌2-烯酸还原酶的表达系统的可用性将促进这种不寻常的“烯”-还原酶类的未来表征，并扩展可用于碳-碳双键的对映选择性氢化的生物催化工具箱。
‘Ene’-reductases have attracted significant attention for the preparation of chemical intermediates and biologically active products. To date, research has been focussed primarily on Old Yellow Enzyme-like proteins, due to their ease of handling, whereas 2-enoate reductases from clostridia have received much less attention, because of their oxygen sensitivity and a lack of suitable expression systems. A hypothetical 2-enoate reductase gene, fldZ, was identified in Clostridium sporogenes DSM 795. The encoded protein shares a high degree of homology to clostridial FMN- and FAD-dependent 2-enoate reductases, including the cinnamic acid reductase proposed to be involved in amino acid metabolism in proteolytic clostridia. The gene was cloned and overexpressed in Escherichia coli. Successful expression depended on the use of strictly anaerobic conditions for both growth and enzyme preparation, since FldZ was oxygen-sensitive. The enzyme reduced aromatic enoates, such as cinnamic acid or p-coumaric acid, but not short chain unsaturated aliphatic acids. The β,β-disubstituted nitroalkene, (E)-1-nitro-2-phenylpropene, was reduced to enantiopure (R)-1-nitro-2-phenylpropane with a yield of 90 %. By contrast, the α,β-disubstituted nitroalkene, (E)-2-nitro-1-phenylpropene, was reduced with a moderate yield of 56 % and poor enantioselectivity (16 % ee for (S)-2-nitro-1-phenylpropane). The availability of an expression system for this recombinant clostridial 2-enoate reductase will facilitate future characterisation of this unusual class of ‘ene’-reductases, and expand the biocatalytic toolbox available for enantioselective hydrogenation of carbon-carbon double bonds.
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影响因子：
5.4
作者：
Hall, Melanie;Stueckler, Clemens;Faber, Kurt
通讯作者：
Faber, Kurt
影响因子：
4.1
作者：
Kim, Nag-Jong;Choi, Jong Hyun;Lee, Pyung Cheon
通讯作者：
Lee, Pyung Cheon
影响因子：
3.5
作者：
GIESEL, H;MACHACEK, G;SIMON, H
通讯作者：
SIMON, H
影响因子：
2.1
作者：
Li, H;Williams, P;Stephens, G
通讯作者：
Stephens, G
DOI：
10.1515/bchm2.1982.363.1.609
发表时间：
1982-01-01
影响因子：
--
作者：
BUHLER, M;SIMON, H
通讯作者：
SIMON, H