The branched chain keto acid dehydrogenase of P. putida is composed of four polypeptides, E1alpha, E1beta, E2 and LPD-val, the, specific E3 component of this complex. The E1 component is important in catalysis since this is the site of regulation of enzyme activity in both P. putida and higher eukaryotes and binds thiamin pyrophosphate. Although the E2 and E3 components have been well studied, little is known about the structure and function of keto acid dehydrogenase E1 components including the binding site for thiamin pyrophosphate. The bkd operon encoding the four proteins of the complex has been isolated, sequenced and found to contain four tightly linked genes. The bkd genes have been subcloned and expressed in E. coli establishing the gene order and direction of transcription. Combining extracts containing the expressed proteins gives enzyme activity and provides the basis for assays for each component. Recently, the upstream region was found to contain a gene encoding a protein with 37.5 % amino acid identity to the leucine responsive protein (LRP) of E. coli. Evidence from plasmid constructions containing this gene and in vitro mutagenesis indicate that the lrp-like gene is involved in the positive expression of the bkd operon. Methylmalonate semialdehyde dehydrogenase, the final enzyme in the valine catabolic pathway, was characterized in our laboratory several years ago and has recently been found in mammals. The mms operon containing the methylmalonate semialdehyde dehydrogenase structural gene has been cloned and sequenced from P. aeruginosa PAO. The mms operon also includes the gene for 3-hydroxyisobutyrate dehydrogenase, the enzyme immediately preceding methylmalonate semialdehyde dehydrogenase in the valine catabolic pathway. A gene, mmsR, encoding a regulatory protein with similarity to AraC, is immediately upstream. Inactivation of mms also inactivated the other two genes, but the mutants were still able to grow on valine agar suggesting an alternate pathway for valine metabolism. The specific aims of this research are to: 1) Study the structure and function of E1alpha-beta from P. putida branched chain keto acid dehydrogenase. 2) Study the action of LRP in expression of the bkd operon. 3) Study the regulation of the mms operon. 4) Look for alternate pathways of valine metabolism in P. aeruginosa.
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