New Thermophilic α/β Class Epoxide Hydrolases Found in Metagenomes From Hot Environments.
New Thermophilic α/β Class Epoxide Hydrolases Found in Metagenomes From Hot Environments.
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DOI：
10.3389/fbioe.2018.00144
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发表时间：
2018
影响因子：
5.7
通讯作者：
Monti D
中科院分区：
文献类型：
--
作者：
Ferrandi EE;Sayer C;De Rose SA;Guazzelli E;Marchesi C;Saneei V;Isupov MN;Littlechild JA;Monti D
在俄罗斯和中国的温泉样品宏基因组中分别鉴定出两种新的环氧化物水解酶Sibe-EH和CH 65-EH。克隆了两种α/β水解酶超家族折叠酶，在大肠杆菌中高效表达，纯化并鉴定了其结构。新的EH对广泛的底物具有活性，特别是Sibe-EH在顺式-2，3-环氧丁烷的去对称化中具有优异的性能，产生ee超过99%的（2 R，3R）-二醇产物。有趣的是，这些酶还水解（4 R）-柠檬烯-1，2-环氧化物，其中Sibe-EH对反式异构体具有特异性。Sibe-EH是溶液中的单体，而CH 65-EH是二聚体。两种酶都显示出高的解链温度，其中CH 65-EH在85°C下最高，在70°C下热处理3小时后保留其初始活性的80%，使其成为所述的最耐热的野生型环氧化物水解酶。Sibe-EH和CH_（65）-EH已结晶，其结构分别测定到高分辨率，1.6和1.4 μ m。CH 65-EH酶通过其帽结构域形成二聚体，与先前描述的EH相比，具有不同的单体相对取向。活性位点空腔的入口位于与其他已知细菌和哺乳动物EH相关的CH 65-EH和Sibe-EH中的不同位置。
Two novel epoxide hydrolases (EHs), Sibe-EH and CH65-EH, were identified in the metagenomes of samples collected in hot springs in Russia and China, respectively. The two α/β hydrolase superfamily fold enzymes were cloned, over-expressed in Escherichia coli, purified and characterized. The new EHs were active toward a broad range of substrates, and in particular, Sibe-EH was excellent in the desymmetrization of cis-2,3-epoxybutane producing the (2R,3R)-diol product with ee exceeding 99%. Interestingly these enzymes also hydrolyse (4R)-limonene-1,2-epoxide with Sibe-EH being specific for the trans isomer. The Sibe-EH is a monomer in solution whereas the CH65-EH is a dimer. Both enzymes showed high melting temperatures with the CH65-EH being the highest at 85°C retaining 80% of its initial activity after 3 h thermal treatment at 70°C making it the most thermal tolerant wild type epoxide hydrolase described. The Sibe-EH and CH65-EH have been crystallized and their structures determined to high resolution, 1.6 and 1.4 Å, respectively. The CH65-EH enzyme forms a dimer via its cap domains with different relative orientation of the monomers compared to previously described EHs. The entrance to the active site cavity is located in a different position in CH65-EH and Sibe-EH in relation to other known bacterial and mammalian EHs.