BIOCHEMISTRY AND GENETICS OF LIPOAMIDE DEHYDROGENASE

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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.