The long term objective of this research is to study the biochemistry and genetics of lipoamide dehydrogenases in pseudomonads which are unique in that they produce two structurally and functionally distinct lipoamide dehydrogenases. Mammals produce a single lipoamide dehydrogenase which serves pyruvate, 2-ketoglutarate and branched chain keto acid dehydrogenase. In man, genetic deficiency of lipoamide dehydrogenase results in lactic acidosis in newborns which is manifested by rapid neurological deterioration and an early death. Escherichia coli produces a single lipoamide dehydrogenase which is the E3 subunit of pyruvate and 2-ketoglutarate dehydrogenases. In contrast, both P. putida and P. aeruginosa produce two lipoamide dehydrogenases, LPD-glc and LPD-val. LPD-glc is the only lipoamide dehydrogenase produce during growth on glucose synthetic medium and is the E3 subunit of 2-ketoglutarate dehydrogenase and the L-factor in glycine oxidation. LPD-val is produced during growth in valine synthetic medium and is the specific E3 subunit of branched chain keto acid dehydrogenase. Recent data raised the possibility that there is also a separate lipoamide dehydrogenase for pyruvate dehydrogenase in P. putida. E. coli and pig heart lipoamide dehydrogenases, human glutathione reductase and mercuric reductase are all redox-active disulfide flavoproteins and share significant regions of homology suggesting a common evolutionary pathway for these apparently disparate enzymes. Studies based on amino acid compositions suggest the LPD-glc is related to E. coli and pig heart lipoamide dehydrogenases, but not to LPD-val. LPD-val appears to be unrelated to any of the other redox-active disulfide flavoproteins, except possibly mercuric reductase.
The Specific Aims of this study are to 1: Determine the primary structure of LPD-val and LPD-glc. The objective of this research will be to determine the evolutionary relationships between these two proteins and other redox-active disulfide flavoproteins. 2. Purify pyruvate dehydrogenase from P. putida to determine if there is a separate structural gene for this complex.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM030428-06
Application #
3278195
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1982-07-01
Project End
1989-08-31
Budget Start
1987-09-01
Budget End
1988-08-31
Support Year
6
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Oklahoma Health Sciences Center
Department
Type
School of Medicine & Dentistry
DUNS #
937727907
City
Oklahoma City
State
OK
Country
United States
Zip Code
73117
Karp, Xantha; Ambros, Victor (2012) Dauer larva quiescence alters the circuitry of microRNA pathways regulating cell fate progression in C. elegans. Development 139:2177-86
Karp, Xantha; Hammell, Molly; Ow, Maria C et al. (2011) Effect of life history on microRNA expression during C. elegans development. RNA 17:639-51
Karp, Xantha; Ambros, Victor (2011) The developmental timing regulator HBL-1 modulates the dauer formation decision in Caenorhabditis elegans. Genetics 187:345-53
Hammell, Christopher M; Lubin, Isabella; Boag, Peter R et al. (2009) nhl-2 Modulates microRNA activity in Caenorhabditis elegans. Cell 136:926-38
Hammell, Christopher M; Karp, Xantha; Ambros, Victor (2009) A feedback circuit involving let-7-family miRNAs and DAF-12 integrates environmental signals and developmental timing in Caenorhabditis elegans. Proc Natl Acad Sci U S A 106:18668-73
Luo, J; Burns, G; Sokatch, J R (1993) Construction of chromosomal recA mutants of Pseudomonas putida PpG2. Gene 136:263-6
Mattevi, A; Obmolova, G; Sokatch, J R et al. (1992) The refined crystal structure of Pseudomonas putida lipoamide dehydrogenase complexed with NAD+ at 2.45 A resolution. Proteins 13:336-51
Palmer, J A; Hatter, K; Sokatch, J R (1991) Cloning and sequence analysis of the LPD-glc structural gene of Pseudomonas putida. J Bacteriol 173:3109-16
Palmer, J A; Madhusudhan, K T; Hatter, K et al. (1991) Cloning, sequence and transcriptional analysis of the structural gene for LPD-3, the third lipoamide dehydrogenase of Pseudomonas putida. Eur J Biochem 202:231-40
Burns, G; Brown, T; Hatter, K et al. (1989) Sequence analysis of the lpdV gene for lipoamide dehydrogenase of branched-chain-oxoacid dehydrogenase of Pseudomonas putida. Eur J Biochem 179:61-9

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