Abstract

The existence of three lipoamide dehydrogenases in pseudomonads provides a unique opportunity to study structure, function and evolution in redox-active disulfide flavoproteins. LPD-val is absolutely specific for branched chain keto acid dehydrogenase and LPD-glc functions only with pyruvate and 2-ketoglutarate dehydrogenases. The third lipoamide dehydrogenase, LPD-3, can replace LPD-glc in the pyruvate and 2-ketoglutarate dehydrogenase complexes, but its primary function is unknown. This situation provides the opportunity to study the structure of lipoamide dehydrogenases with a view to identifying functional domains. Study of lipoamide dehydrogenases also has medical importance since genetic defects in this enzyme are one of the causes of lactic acidosis, a severe and usually fatal genetic disease.
The Specific Aims of this research are to: 1.Determine the role of LPD-3. This will accomplished by completing the nucleotide sequence of the region downstream of lpd3, by creating site-directed mutations in lpd3 and by studying the induction of LPD-3 in both wild-type and mutant strains of P. putida. 2.Determine the role of LPD-glc. This will be accomplished by completing the nucleotide sequence upstream of lpdG, by creating site-directed mutations in lpdG and by a transcriptional analysis of lpdG expression. 3.Determine the relationships of LPD-val LPD-glc and LPD-3. We will study this specific aim by creating a strain of P. putida with site-directed mutations in lpdV, lpdG and lpd3 to see what effect this has on enzyme induction and growth. 4. Study the structure and function of LPD-val. W.G.J. Hol at the University of Groningen will attempt to crystallize LPD-val. Knowing the crystal structure, it will be possible to create a series of mutations in LPD-val whose effects can be interpreted. 5.Study the evolutionary relationships of LPD-3 LPD-glc and LPD-val. This will be accomplished by analyzing the primary amino acid sequences of these proteins using PHYLIP programs.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM030428-09
Application #
3278197
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1982-07-01
Project End
1994-11-30
Budget Start
1991-12-01
Budget End
1992-11-30
Support Year
9
Fiscal Year
1992
Total Cost
Indirect Cost

  Institution

Name
University of Oklahoma Health Sciences Center
Department
Type
Schools of Dentistry
DUNS #
937727907
City
Oklahoma City
State
OK
Country
United States
Zip Code
73117

  Publications

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; 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
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
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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