Resolution of branched-chain oxo acid dehydrogenase complex of Pseudomonas aeruginosa PAO.
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.
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism; D007658:Ketone Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013379:Substrate Specificity; D008063:Thioctic Acid / Q000031:analogs & derivatives
D000596:Amino Acids; D009097:Multienzyme Complexes; D010446:Peptide Fragments; C007409:dihydrolipoamide; D008063:Thioctic Acid; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D008058:Dihydrolipoamide Dehydrogenase