Isolation of a third lipoamide dehydrogenase from Pseudomonas putida.
Pseudomonads are the only organisms so far known to produce two lipoamide dehydrogenases (LPDs), LPD-Val and LPD-Glc. LPD-Val is the specific E3 component of branched-chain oxoacid dehydrogenase, and LPD-Glc is the E3 component of 2-ketoglutarate and possibly pyruvate dehydrogenases and the L-factor of the glycine oxidation system. Three mutants of Pseudomonas putida, JS348, JS350, and JS351, affected in lpdG, the gene encoding LPD-Glc, have been isolated; all lacked 2-ketoglutarate dehydrogenase, but two, JS348 and JS351, had normal pyruvate dehydrogenase activity. The pyruvate and 2-ketoglutarate dehydrogenases of the wild-type strain of P. putida were both inhibited by anti-LPD-Glc, but the pyruvate dehydrogenase of the lpdG mutants was not inhibited, suggesting that the mutant pyruvate dehydrogenase E3 component was different from that of the wild type. The lipoamide dehydrogenase present in one of the lpdG mutants, JS348, was isolated and characterized. This lipoamide dehydrogenase, provisionally named LPD-3, differed in molecular weight, amino acid composition, and N-terminal amino acid sequence from LPD-Glc and LPD-Val. LPD-3 was clearly a lipoamide dehydrogenase as opposed to a mercuric reductase or glutathione reductase. LPD-3 was about 60% as effective as LPD-Glc in restoring 2-ketoglutarate dehydrogenase activity and completely restored pyruvate dehydrogenase activity in JS350. These results suggest that LPD-3 is a lipoamide dehydrogenase associated with an unknown multienzyme complex which can replace LPD-Glc as the E3 component of pyruvate and 2-ketoglutarate dehydrogenases in lpdG mutants.
Burns
G
G
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City 73190.
Sykes
P J
PJ
Hatter
K
K
Sokatch
J R
JR
D000595:Amino Acid Sequence; D008058:Dihydrolipoamide Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D007655:Ketoglutarate Dehydrogenase Complex / Q000378:metabolism; D008969:Molecular Sequence Data; D011549:Pseudomonas / Q000201:enzymology*; D013045:Species Specificity
D007655:Ketoglutarate Dehydrogenase Complex; D008058:Dihydrolipoamide Dehydrogenase