Resolution of branched-chain oxo acid dehydrogenase complex of Pseudomonas aeruginosa PAO.
Resolution of branched-chain oxo acid dehydrogenase complex of Pseudomonas aeruginosa PAO.
复制标题
铜绿假单胞菌 PAO 支链含氧酸脱氢酶复合物的分离。
DOI：
10.1042/bj2330737
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发表时间：
1986
期刊：
影响因子：
--
通讯作者：
Sokatch,JR
中科院分区：
文献类型：
--
作者：
McCully,V;Burns,G;Sokatch,JR
从铜绿假单胞菌菌株PAO中纯化支链含氧酸脱氢酶，目的是将复合物分解成其亚基。纯化的复合物由四种蛋白质组成，Mr为36，000，42，000，49，000和50，000。通过热处理将复合物分解为49，000和50，000-Mr蛋白质，通过DEAE-Sepharose色谱法将其分离。49，000-Mr蛋白质通过其催化与多种底物的转酰作用的能力被鉴定为E2亚基，其中二氢硫辛酰胺作为受体。铜绿假单胞菌与恶臭假单胞菌一样，产生两种硫辛酰胺脱氢酶。一种是50，000-Mr蛋白，被鉴定为支链含氧酸脱氢酶的特异性E3亚基，与恶臭假单胞菌的硫辛酰胺脱氢酶LPD-val具有许多共同的性质。第二硫辛酰胺脱氢酶具有Mr 54，000并且对应于恶臭假单胞菌的硫辛酰胺脱氢酶LPD-glc。来自恶臭假单胞菌和铜绿假单胞菌的LPD-val的C-末端CNBr肽的片段紧密对应，在31个氨基酸中仅有两个氨基酸差异。与大肠杆菌硫辛酰胺脱氢酶C-末端的相应片段也具有广泛的同源性。所有三种肽都具有8个氨基酸的共同片段，序列为TIHAHPTL。这种同源性在Dayhoff数据库中的任何其他黄素蛋白中都不明显，这表明该序列可能是硫辛酰胺脱氢酶的特征。
Branched-chain oxo acid dehydrogenase was purified from Pseudomonas aeruginosa strain PAO with the objective of resolving the complex into its subunits. The purified complex consisted of four proteins, of Mr 36,000, 42,000, 49,000 and 50,000. The complex was resolved by heat treatment into the 49,000 and 50,000-Mr proteins, which were separated by chromatography on DEAE-Sepharose. The 49,000-Mr protein was identified as the E2 subunit by its ability to catalyse transacylation with a variety of substrates, with dihydrolipoamide as the acceptor. P. aeruginosa, like P. putida, produces two lipoamide dehydrogenases. One, the 50,000-Mr protein, was identified as the specific E3 subunit of branched-chain oxo acid dehydrogenase and had many properties in common with the lipoamide dehydrogenase LPD-val of P. putida. The second lipoamide dehydrogenase had Mr 54,000 and corresponded to the lipoamide dehydrogenase LPD-glc of P. putida. Fragments of C-terminal CNBr peptides of LPD-val from P. putida and P. aeruginosa corresponded closely, with only two amino acid differences over 31 amino acids. A corresponding fragment at the C-terminal end of lipoamide dehydrogenase from Escherichia coli also showed extensive homology. All three peptides had a common segment of eight amino acids, with the sequence TIHAHPTL. This homology was not evident in any other flavoproteins in the Dayhoff data base which suggests that this sequence might be characteristic of lipoamide dehydrogenase.