6-Oxocyclohex-1-ene-1-carbonyl-coenzyme A hydrolases from obligately anaerobic bacteria: characterization and identification of its gene as a functional marker for aromatic compounds degrading anaerobes
6-Oxocyclohex-1-ene-1-carbonyl-coenzyme A hydrolases from obligately anaerobic bacteria: characterization and identification of its gene as a functional marker for aromatic compounds degrading anaerobes
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DOI：
10.1111/j.1462-2920.2008.01570.x
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发表时间：
2008-06-01
影响因子：
5.1
通讯作者：
Boll, Matthias
中科院分区：
文献类型：
--
作者：
Kuntze, Kevin;Shinoda, Yoshifumi;Boll, Matthias
在厌氧细菌中，大多数芳香族生长底物被引导至苯甲酰辅酶 A (CoA) 降解途径，其中芳香环被脱芳构化并裂解成脂肪族硫羟酸酯。该途径的第一步是由苯甲酰辅酶A还原酶脱芳构化催化，产生双电子还原产物环己-1,5-二烯-1-羰基-辅酶A，随后通过水合酶向其中添加水。接下来的两个步骤迄今为止仅在兼性厌氧菌中进行了研究，包括将 6-羟基氧化为 6-氧代环己-1-烯-1-羰基-CoA (6-OCH-CoA)，添加水和水解环裂解产生 3-羟基庚二基-CoA。在这项工作中，来自专性厌氧细菌金属还原地杆菌 (bamA(Geo)) 和嗜酸合成营养菌 (bamA(Syn)) 的两个苯甲酸盐诱导基因在大肠杆菌中异源表达，纯化并表征为 6-OCH-CoA 水解酶。两种酶均由单个 43 kDa 亚基组成。介绍了这些酶的一些特性，并与兼性厌氧菌的同系物进行了比较。将 bamA(Geo) 和 bamA(Syn) 的核苷酸序列与兼性厌氧菌的相应基因进行比对，鉴定出高度保守的 DNA 区域，从而能够区分编码 6-OCH-CoA 水解酶的基因和编码相关酶的基因。从保守区域推导出简并寡核苷酸引物对，并将其应用于聚合酶链式反应。使用这些引物，从几乎所有已知的使用芳香族生长底物的兼性和专性厌氧菌的 DNA 中特异性扩增出 6-OCH-CoA 水解酶基因的预期 DNA 片段。唯一的例外是芳香化合物降解沼泽红假单胞菌，它独特地使用改良的苯甲酰辅酶A降解途径。使用寡核苷酸引物，预期的 DNA 片段也在甲苯降解和间二甲苯降解富集培养物中扩增，证明了其在不太明确的细菌群落中的潜在用途。这项工作中建立的基因探针首次为检测芳香族化合物降解厌氧菌的中心功能提供了通用工具。
In anaerobic bacteria, most aromatic growth substrates are channelled into the benzoyl-coenzyme A (CoA) degradation pathway where the aromatic ring is dearomatized and cleaved into an aliphatic thiol ester. The initial step of this pathway is catalysed by dearomatizing benzoyl-CoA reductases yielding the two electron-reduction product, cyclohexa-1,5-diene-1-carbonyl-CoA, to which water is subsequently added by a hydratase. The next two steps have so far only been studied in facultative anaerobes and comprise the oxidation of the 6-hydroxyl-group to 6-oxocyclohex-1-ene-1-carbonyl-CoA (6-OCH-CoA), the addition of water and hydrolytic ring cleavage yielding 3-hydroxypimelyl-CoA. In this work, two benzoate-induced genes from the obligately anaerobic bacteria, Geobacter metallireducens (bamA(Geo)) and Syntrophus aciditrophicus (bamA(Syn)), were heterologously expressed in Escherichia coli, purified and characterized as 6-OCH-CoA hydrolases. Both enzymes consisted of a single 43 kDa subunit. Some properties of the enzymes are presented and compared with homologues from facultative anaerobes. An alignment of the nucleotide sequences of bamA(Geo) and bamA(Syn) with the corresponding genes from facultative anaerobes identified highly conserved DNA regions, which enabled the discrimination of genes coding for 6-OCH-CoA hydrolases from those coding for related enzymes. A degenerate oligonucleotide primer pair was deduced from conserved regions and applied in polymerase chain reaction reactions. Using these primers, the expected DNA fragment of the 6-OCH-CoA hydrolase genes was specifically amplified from the DNA of nearly all known facultative and obligate anaerobes that use aromatic growth substrates. The only exception was the aromatic compound-degrading Rhodopseudomonas palustris, which uniquely uses a modified benzoyl-CoA degradation pathway. Using the oligonucleotide primers, the expected DNA fragment was also amplified in a toluene-degrading and a m-xylene-degrading enrichment culture demonstrating its potential use in less defined bacterial communities. The gene probe established in this work provides for the first time a general tool for the detection of a central functionality in aromatic compound-degrading anaerobes.