Cloning, sequence and transcriptional analysis of the structural gene for LPD-3, the third lipoamide dehydrogenase of Pseudomonas putida.
Cloning, sequence and transcriptional analysis of the structural gene for LPD-3, the third lipoamide dehydrogenase of Pseudomonas putida.
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恶臭假单胞菌第三种硫辛酰胺脱氢酶LPD-3结构基因的克隆、序列和转录分析。
DOI：
10.1111/j.1432-1033.1991.tb16367.x
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发表时间：
1991
期刊：
影响因子：
--
通讯作者：
Sokatch,JR
中科院分区：
文献类型：
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作者：
Palmer,JA;Madhusudhan,KT;Hatter,K;Sokatch,JR
从恶臭假单胞菌（Pseudomonasputida）的硫辛酰胺脱氢酶基因文库中克隆了第三个硫辛酰胺脱氢酶基因lpd 3。putia PpG 2 DNA克隆入大肠杆菌TB 1. lpd 3及其侧翼区的核苷酸序列表明lpd 3不是操纵子的一部分，而操纵子是原核硫辛酰胺脱氢酶所特有的。在转录起始点上游207个碱基处发现了一个开放阅读框，但它编码在相反的pd 3链上。没有证据表明lpd 3下游有一个开放的阅读帧。flpd 3基因编码区全长1401 bp，含466个氨基酸和一个终止密码子，G/C含量为62.4%。转录起始位点位于翻译起始点上游33 bp处。第三个硫辛酰胺脱氢酶（LPD-3）与P. putida，45%与2-酮戊二酸脱氢酶和丙酮酸多酶复合物，45.9%与支链含氧酸复合物的硫辛酰胺脱氢酶。LPD-3与真核硫辛酰胺脱氢酶更密切相关，因为它与猪和人硫辛酰胺脱氢酶具有53.6%的氨基酸序列同一性，与酵母硫辛酰胺脱氢酶具有51.1%的同一性。LPD-3在野生型P中不产生。putidaPpG 2在各种生长条件下的生长。然而，LPD-3在P中产生。putidaPpG 2携带pSP 14，pSP 14是一种基于pKT 240的克隆，具有完整的relpd 3基因加上前导序列的104个碱基。LPD-3在P中的唯一作用。putida是2-酮戊二酸脱氢酶和丙酮酸多酶复合物的硫辛酰胺脱氢酶的替代物，当后者无活性或缺失时。
The third lipoamide dehydrogenase structural gene ofPseudomonas putida, lpd3, was isolated from a library ofP. putidaPpG2 DNA cloned inEscherichia coliTB1. The nucleotide sequence oflpd3and its flanking regions indicate thatlpd3is not part of an operon, which is unique for a prokaryotic lipoamide dehydrogenase. An open reading frame was found 207 bases upstream from the start of transcription, but is encoded on the strand oppositelpd3. There is no evidence of an open reading frame immediately downstream fromlpd3. The coding region oflpd3consists of 1401 bp, providing for 466 amino acids plus a stop codon with a G/C content of 62.4%. The transcriptional start site was located 33‐bp upstream from the start of translation. The third lipoamide dehydrogenase (LPD‐3) shares amino acid identity with the other two lipoamide dehydrogenases ofP. putida, 45% with that of the 2‐oxoglutarate dehydrogenase and pyruvate multienzyme complexes, and 45.9% with the lipoamide dehydrogenase of the branched‐chain oxoacid complex. LPD‐3 is more closely related to eukaryotic lipoamide dehydrogenases since it has 53.6% amino acid sequence identity with pig and human lipoamide dehydrogenases and 51.1% identity with yeast lipoamide dehydrogenase. LPD‐3 was not produced in wild‐typeP. putidaPpG2 under a variety of growth conditions. However, LPD‐3 was produced inP. putidaPpG2 carrying pSP14, a pKT240‐based clone with the entirelpd3gene plus 104 bases of the leader. The only demonstrated role of LPD‐3 inP. putidais a substitute for lipoamide dehydrogenase of the 2‐oxoglutarate dehydrogenase and pyruvate multienzyme complexes when the latter is inactive or missing.