A catalytically versatile benzoyl-CoA reductase, key enzyme in the degradation of methyl- and halobenzoates in denitrifying bacteria
A catalytically versatile benzoyl-CoA reductase, key enzyme in the degradation of methyl- and halobenzoates in denitrifying bacteria
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DOI：
10.1074/jbc.ra118.003329
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发表时间：
2018-05
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影响因子：
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通讯作者：
Oliver Tiedt;Jonathan M Fuchs;W. Eisenreich;M. Boll
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作者：
Oliver Tiedt;Jonathan M Fuchs;W. Eisenreich;M. Boll
I类苯甲酰辅酶A（BzCoA）还原酶（BCR）是芳香族化合物厌氧降解的关键酶。它们可能通过基于自由基的Birch样还原机制催化中心BzCoA中间体及其类似物的ATP依赖性还原为共轭环状1，5-二烯酰基-CoA。这种酶于1995年被发现，迄今为止，它仍然是唯一一种分离的、生物化学上可获得的BCR，这主要是因为BCR非常不稳定，而且它们的异源生产迄今为止基本上失败了。在这里，我们描述了一个平台，在大肠杆菌中编码指定的3-甲基苯甲酰基-CoA还原酶的四个结构基因的异源表达从相关的微生物物种Thauera chlorobenzoica（MBRTcl）。这种还原酶代表了ATP依赖性BCR的一个不同亚类的原型，这些亚类被认为参与甲基取代的BzCoA类似物的降解。重组MBRTcl具有αβγδ亚基结构，含有3个低电位[4Fe-4S]簇，并且是高度氧不稳定的。它催化BzCoA的ATP依赖性还原脱芳构化，其中每两个电子转移2.3-2.8个ATP水解，并且优先脱芳构化Meta位和对位的甲基和氯取代的类似物。NMR分析显示，3-甲基苯甲酰基-CoA被区域选择性地还原为3-甲基-1，5-二烯酰基-CoA。4-氯-BzCoA还原脱氯为BzCoA的前所未有的反应可能是通过从还原中间体中消除HCl而进行的，这使得T.氯苯甲酸对4-氯苯甲酸酯。在这项工作中建立的异源表达平台，使细菌和古细菌BCR和BCR样自由基酶的生产，分离和表征，其中许多功能仍然未知。
Class I benzoyl-CoA (BzCoA) reductases (BCRs) are key enzymes in the anaerobic degradation of aromatic compounds. They catalyze the ATP-dependent reduction of the central BzCoA intermediate and analogues of it to conjugated cyclic 1,5-dienoyl-CoAs probably by a radical-based, Birch-like reduction mechanism. Discovered in 1995, the enzyme from the denitrifying bacterium Thauera aromatica (BCRTar) has so far remained the only isolated and biochemically accessible BCR, mainly because BCRs are extremely labile, and their heterologous production has largely failed so far. Here, we describe a platform for the heterologous expression of the four structural genes encoding a designated 3-methylbenzoyl-CoA reductase from the related denitrifying species Thauera chlorobenzoica (MBRTcl) in Escherichia coli. This reductase represents the prototype of a distinct subclass of ATP-dependent BCRs that were proposed to be involved in the degradation of methyl-substituted BzCoA analogues. The recombinant MBRTcl had an αβγδ-subunit architecture, contained three low-potential [4Fe-4S] clusters, and was highly oxygen-labile. It catalyzed the ATP-dependent reductive dearomatization of BzCoA with 2.3–2.8 ATPs hydrolyzed per two electrons transferred and preferentially dearomatized methyl- and chloro-substituted analogues in meta- and para-positions. NMR analyses revealed that 3-methylbenzoyl-CoA is regioselectively reduced to 3-methyl-1,5-dienoyl-CoA. The unprecedented reductive dechlorination of 4-chloro-BzCoA to BzCoA probably via HCl elimination from a reduced intermediate allowed for the previously unreported growth of T. chlorobenzoica on 4-chlorobenzoate. The heterologous expression platform established in this work enables the production, isolation, and characterization of bacterial and archaeal BCR and BCR-like radical enzymes, for many of which the function has remained unknown.