Anaerobic toluene catabolism of Thauera aromatica:: the bbs operon codes for enzymes of β oxidation of the intermediate benzylsuccinate
Anaerobic toluene catabolism of Thauera aromatica:: the bbs operon codes for enzymes of β oxidation of the intermediate benzylsuccinate
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DOI：
10.1128/jb.182.2.272-277.2000
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发表时间：
2000-01-01
影响因子：
3.2
通讯作者：
Heider, J
中科院分区：
文献类型：
--
作者：
Leuthner, B;Heider, J
甲苯厌氧氧化为苯甲酰辅酶A（苯甲酰-CoA）的途径包括由苄基琥珀酸合成酶催化的初始反应，将甲苯的甲基添加到富马酸酯共底物的双键上的甘氨酰自由基酶，以及随后的苄基琥珀酸的β-氧化途径。苄基琥珀酸合酶已被详细研究，而参与苄基琥珀酸的β氧化的酶是未知的。Ne通过分析甲苯培养细胞中底物诱导的蛋白质研究了这些酶。甲苯诱导的蛋白质进行了鉴定和N-末端测序。这些蛋白质中的九个由8.5 kb操纵子编码，该操纵子由bbs（苄基琥珀酸的β-氧化）基因组成，其产物显然参与苄基琥珀酸的β-氧化途径。其中两个基因bbsE和bbsF编码琥珀酰辅酶A的亚基：苄基琥珀酸辅酶A转移酶，其活性先前在甲苯生长的Thauera aromatica中检测到。bbsG基因编码特定的苄基琥珀酰辅酶A脱氢酶，如通过在大肠杆菌中过表达该基因和检测酶活性所证实的。该途径的其他酶可能由bbsH（烯酰辅酶A水合酶）、bbsCD（3-羟酰辅酶A脱氢酶）和bbsB（3-氧代酰基辅酶A硫解酶）编码。操纵子含有两个额外的基因bbsA和bbsI，它们没有明显的功能。bbs操纵子仅在甲苯培养的细胞中表达，并在转录水平上受到调控。启动子定位揭示了bbsA基因上游的转录起始位点。这代表了Thauera spp中第一个已知的启动子位点。
The pathway of anaerobic toluene oxidation to benzoyl coenzyme A (benzoyl-CoA) consists of an initial reaction catalyzed by benzylsuccinate synthase, a glycyl radical enzyme adding the methyl group of toluene to the double bond of a fumarate cosubstrate, and a subsequent beta-oxidation pathway of benzylsuccinate. Benzylsuccinate synthase has been studied in some detail, whereas the enzymes participating in beta oxidation of benzylsuccinate are unknown. Ne have investigated these enzymes by analyzing substrate-induced proteins in toluene-grown cells. Toluene-induced proteins were identified and N-terminally sequenced. Nine of these proteins are encoded by an 8.5-kb operon consisting of bbs (beta-oxidation of benzylsuccinate) genes whose products are apparently involved in the beta-oxidation pathway of benzylsuccinate. Two of the genes, bbsE and bbsF, code for the subunits of a succinyl-CoA:benzylsuccinate CoA-transferase whose activity was previously detected in toluene-grown Thauera aromatica. The bbsG gene codes for a specific benzylsuccinyl-CoA dehydrogenase, as confirmed by overexpression of the gene in Escherichia coli and detection of enzyme activity. The further enzymes of the pathway are probably encoded by bbsH (enoyl-CoA hydratase), bbsCD (3-hydroxyacyl-CoA dehydrogenase), and bbsB (3-oxoacyl-CoA thiolase), The operon contains two additional genes, bbsA and bbsI, for which no obvious function could be derived. The bbs operon is expressed only in toluene-grown cells and is regulated at the transcriptional level. Promoter mapping revealed a transcription start site upstream of the bbsA gene. This represents the first known promoter site in Thauera spp.