Palmer J A, Madhusudhan K T, Hatter K, Sokatch J R
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City 73190.
Eur J Biochem. 1991 Dec 5;202(2):231-40. doi: 10.1111/j.1432-1033.1991.tb16367.x.
The third lipoamide dehydrogenase structural gene of Pseudomonas putida, lpd3, was isolated from a library of P. putida PpG2 DNA cloned in Escherichia coli TB1. The nucleotide sequence of lpd3 and its flanking regions indicate that lpd3 is 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 opposite lpd3. There is no evidence of an open reading frame immediately downstream from lpd3. The coding region of lpd3 consists 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 of P. 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-type P. putida PpG2 under a variety of growth conditions. However, LPD-3 was produced in P. putida PpG2 carrying pSP14, a pKT240-based clone with the entire lpd3 gene plus 104 bases of the leader. The only demonstrated role of LPD-3 in P. putida is as a substitute for lipoamide dehydrogenase of the 2-oxoglutarate dehydrogenase and pyruvate multienzyme complexes when the latter is inactive or missing.
恶臭假单胞菌的第三个硫辛酰胺脱氢酶结构基因lpd3，是从克隆于大肠杆菌TB1的恶臭假单胞菌PpG2 DNA文库中分离得到的。lpd3及其侧翼区域的核苷酸序列表明，lpd3不是操纵子的一部分，这对于原核硫辛酰胺脱氢酶来说是独特的。在转录起始点上游207个碱基处发现了一个开放阅读框，但它位于与lpd3相反的链上。在lpd3紧下游没有开放阅读框的证据。lpd3的编码区由1401个碱基对组成，编码466个氨基酸加一个终止密码子，G/C含量为62.4%。转录起始位点位于翻译起始点上游33个碱基处。第三个硫辛酰胺脱氢酶（LPD - 3）与恶臭假单胞菌的其他两个硫辛酰胺脱氢酶具有氨基酸同一性，与2 - 氧代戊二酸脱氢酶和丙酮酸多酶复合物的硫辛酰胺脱氢酶同一性为45%，与支链氧代酸复合物的硫辛酰胺脱氢酶同一性为45.9%。LPD - 3与真核硫辛酰胺脱氢酶的关系更为密切，因为它与猪和人的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。在多种生长条件下，野生型恶臭假单胞菌PpG2中不产生LPD - 3。然而，在携带pSP14的恶臭假单胞菌PpG2中产生了LPD - 3，pSP14是一个基于pKT240的克隆，带有完整的lpd3基因加104个碱基的前导序列。LPD - 3在恶臭假单胞菌中唯一已证实的作用是，当2 - 氧代戊二酸脱氢酶和丙酮酸多酶复合物的硫辛酰胺脱氢酶无活性或缺失时，替代其发挥作用。
