Cloning, sequence and transcriptional analysis of the structural gene for LPD-3, the third lipoamide dehydrogenase of Pseudomonas putida.
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.
Palmer
J A
JA
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City 73190.
Madhusudhan
K T
KT
Hatter
K
K
Sokatch
J R
JR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D005075:Biological Evolution; D015153:Blotting, Western; D003001:Cloning, Molecular; D003062:Codon; D004269:DNA, Bacterial / Q000235:genetics; D008058:Dihydrolipoamide Dehydrogenase / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D015183:Restriction Mapping; D016415:Sequence Alignment; D014158:Transcription, Genetic
D003062:Codon; D004269:DNA, Bacterial; D007527:Isoenzymes; D008058:Dihydrolipoamide Dehydrogenase; D012194:RNA-Directed DNA Polymerase