Identification and characterization of the tungsten-containing class of benzoyl-coenzyme A reductases
Identification and characterization of the tungsten-containing class of benzoyl-coenzyme A reductases
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DOI：
10.1073/pnas.0905073106
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发表时间：
2009-10-20
影响因子：
11.1
通讯作者：
Boll, Matthias
中科院分区：
文献类型：
--
作者：
Kung, Johannes W.;Loeffler, Claudia;Boll, Matthias
芳香族化合物在自然界中分布广泛，只能通过微生物进行生物矿化。在厌氧细菌中，苯甲酰辅酶A (BCoA) 是芳香族降解的中心中间体，并作为脱芳构化 BCoA 还原酶 (BCR) 的底物。在兼性厌氧菌中，BCoA 还原为环己酰基-1,5-二烯酰基-1-羧基-CoA（二烯酰基-CoA）在机械上是困难的，这是由化学计量的 ATP 水解驱动的，由可溶性、含有三个 [4Fe-4S] 簇的 BCR 催化。在这项工作中，建立了专性厌氧金属还原地杆菌的 BCR 体外测定方法。它遵循逆反应，在各种电子受体存在下由二烯酰辅酶A形成BCoA。苯甲酸盐诱导的活性对于二烯酰辅酶A具有高度特异性（K-m=24+/-4μM）。相应的氧敏感酶经多次层析纯化，富集度115倍，收率18%。 185 kDa 酶包含 73 kDa 和 20 kDa 亚基，表明其为 alpha(2)beta(2) 组成。 MS 分析显示这些亚基是苯甲酸盐诱导的 bamBC 基因的产物。 αβ单元含有0.9W、15Fe和12.5酸不稳定硫。 EPR 光谱结果表明每个 αβ 单元存在 1 个 [3Fe-4S](0/+1) 和 3 个 [4Fe-4S](+1/+2) 簇；氧化的 BamBC 表现出 W(V) 物种典型的 EPR 信号。 FeS簇和W-辅因子只能被二烯酰辅酶A完全还原。 BamBC 代表了先前未描述的一类脱芳香化 BCR 的原型，该类 BCR 与兼性厌氧菌的 ATP 依赖性酶完全不同。
Aromatic compounds are widely distributed in nature and can only be biomineralized by microorganisms. In anaerobic bacteria, benzoyl-CoA (BCoA) is a central intermediate of aromatic degradation, and serves as substrate for dearomatizing BCoA reductases (BCRs). In facultative anaerobes, the mechanistically difficult reduction of BCoA to cyclohexa-1,5-dienoyl-1-carboxyl-CoA (dienoyl-CoA) is driven by a stoichiometric ATP hydrolysis, catalyzed by a soluble, three [4Fe-4S] cluster-containing BCR. In this work, an in vitro assay for BCR from the obligately anaerobic Geobacter metallireducens was established. It followed the reverse reaction, the formation of BCoA from dienoyl-CoA in the presence of various electron acceptors. The benzoate-induced activity was highly specific for dienoyl-CoA (K-m = 24 +/- 4 mu M). The corresponding oxygen-sensitive enzyme was purified by several chromatographic steps with a 115-fold enrichment and a yield of 18%. The 185-kDa enzyme comprised 73- and 20-kDa subunits, suggesting an alpha(2)beta(2)-composition. MS analysis revealed the subunits as products of the benzoate-induced bamBC genes. The alpha beta unit contained 0.9 W, 15 Fe, and 12.5 acid-labile sulfur. Results from EPR spectroscopy suggest the presence of one [3Fe-4S](0/+1) and three [4Fe-4S](+1/+2) clusters per alpha beta unit; oxidized BamBC exhibited an EPR signal typical for a W(V) species. The FeS clusters and the W-cofactor could only be fully reduced by dienoyl-CoA. BamBC represents the prototype of a previously undescribed class of dearomatizing BCRs that differ completely from the ATP-dependent enzymes from facultative anaerobes.