Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1989 Feb;171(2):665–668. doi: 10.1128/jb.171.2.665-668.1989

Isolation of a third lipoamide dehydrogenase from Pseudomonas putida.

G Burns 1, P J Sykes 1, K Hatter 1, J R Sokatch 1
PMCID: PMC209648  PMID: 2914869

Abstract

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.

Full text

PDF
665

Images in this article

667Image
on p.667

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barrineau P., Gilbert P., Jackson W. J., Jones C. S., Summers A. O., Wisdom S. The DNA sequence of the mercury resistance operon of the IncFII plasmid NR1. J Mol Appl Genet. 1984;2(6):601–619. [PubMed] [Google Scholar]
  2. Brown N. L., Ford S. J., Pridmore R. D., Fritzinger D. C. Nucleotide sequence of a gene from the Pseudomonas transposon Tn501 encoding mercuric reductase. Biochemistry. 1983 Aug 16;22(17):4089–4095. doi: 10.1021/bi00286a015. [DOI] [PubMed] [Google Scholar]
  3. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed] [Google Scholar]
  4. Delaney R., Burns G., Sokatch J. R. Relationship of lipoamide dehydrogenases from Pseudomonas putida to other FAD-linked dehydrogenases. FEBS Lett. 1984 Mar 26;168(2):265–270. doi: 10.1016/0014-5793(84)80259-8. [DOI] [PubMed] [Google Scholar]
  5. Duncan M. J., Fraenkel D. G. alpha-Ketoglutarate dehydrogenase mutant of Rhizobium meliloti. J Bacteriol. 1979 Jan;137(1):415–419. doi: 10.1128/jb.137.1.415-419.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fothergill J. C., Guest J. R. Catabolism of L-lysine by Pseudomonas aeruginosa. J Gen Microbiol. 1977 Mar;99(1):139–155. doi: 10.1099/00221287-99-1-139. [DOI] [PubMed] [Google Scholar]
  7. Guest J. R. Aspects of the molecular biology of lipoamide dehydrogenase. Adv Neurol. 1978;21:219–244. [PubMed] [Google Scholar]
  8. Marshall V. D., Sokatch J. R. Regulation of valine catabolism in Pseudomonas putida. J Bacteriol. 1972 Jun;110(3):1073–1081. doi: 10.1128/jb.110.3.1073-1081.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. McCully V., Burns G., Sokatch J. R. Resolution of branched-chain oxo acid dehydrogenase complex of Pseudomonas aeruginosa PAO. Biochem J. 1986 Feb 1;233(3):737–742. doi: 10.1042/bj2330737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Misra T. K., Brown N. L., Haberstroh L., Schmidt A., Goddette D., Silver S. Mercuric reductase structural genes from plasmid R100 and transposon Tn501: functional domains of the enzyme. Gene. 1985;34(2-3):253–262. doi: 10.1016/0378-1119(85)90134-9. [DOI] [PubMed] [Google Scholar]
  11. Robinson B. H., Taylor J., Sherwood W. G. Deficiency of dihydrolipoyl dehydrogenase (a component of the pyruvate and alpha-ketoglutarate dehydrogenase complexes): a cause of congenital chronic lactic acidosis in infancy. Pediatr Res. 1977 Dec;11(12):1198–1202. doi: 10.1203/00006450-197712000-00006. [DOI] [PubMed] [Google Scholar]
  12. Sokatch J. R., Burns G. Oxidation of glycine by Pseudomonas putida requires a specific lipoamide dehydrogenase. Arch Biochem Biophys. 1984 Feb 1;228(2):660–666. doi: 10.1016/0003-9861(84)90036-5. [DOI] [PubMed] [Google Scholar]
  13. Sokatch J. R., McCully V., Gebrosky J., Sokatch D. J. Isolation of a specific lipoamide dehydrogenase for a branched-chain keto acid dehydrogenase from Pseudomonas putida. J Bacteriol. 1981 Nov;148(2):639–646. doi: 10.1128/jb.148.2.639-646.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Sokatch J. R., McCully V., Roberts C. M. Purification of a branched-chain keto acid dehydrogenase from Pseudomonas putida. J Bacteriol. 1981 Nov;148(2):647–652. doi: 10.1128/jb.148.2.647-652.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Sokatch J. R., McCully V., Sahm J. G., Reyes-Maguire M. Mutations affecting lipoamide dehydrogenases of Pseudomonas putida. J Bacteriol. 1983 Feb;153(2):969–975. doi: 10.1128/jb.153.2.969-975.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Spencer M. E., Guest J. R. Transcription analysis of the sucAB, aceEF and lpd genes of Escherichia coli. Mol Gen Genet. 1985;200(1):145–154. doi: 10.1007/BF00383328. [DOI] [PubMed] [Google Scholar]
  17. Stephens P. E., Lewis H. M., Darlison M. G., Guest J. R. Nucleotide sequence of the lipoamide dehydrogenase gene of Escherichia coli K12. Eur J Biochem. 1983 Oct 3;135(3):519–527. doi: 10.1111/j.1432-1033.1983.tb07683.x. [DOI] [PubMed] [Google Scholar]
  18. Sykes P. J., Burns G., Menard J., Hatter K., Sokatch J. R. Molecular cloning of genes encoding branched-chain keto acid dehydrogenase of Pseudomonas putida. J Bacteriol. 1987 Apr;169(4):1619–1625. doi: 10.1128/jb.169.4.1619-1625.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sykes P. J., Menard J., McCully V., Sokatch J. R. Conjugative mapping of pyruvate, 2-ketoglutarate, and branched-chain keto acid dehydrogenase genes in Pseudomonas putida mutants. J Bacteriol. 1985 Apr;162(1):203–208. doi: 10.1128/jb.162.1.203-208.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Taylor J., Robinson B. H., Sherwood W. G. A defect in branched-chain amino acid metabolism in a patient with congenital lactic acidosis due to dihydrolipoyl dehydrogenase deficiency. Pediatr Res. 1978 Jan;12(1):60–62. doi: 10.1203/00006450-197801000-00018. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES