The Pyruvate Dehydrogenase Complex of Pseudomonas aeruginosa PAO. Purification, Properties and Characterization of Mutants

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Abstract
Summary: The pyruvate dehydrogenase complex of Pseudomonas aeruginosa PAO was purified by affinity chromatography on ethanol-Sepharose 2B followed by sucrose density gradient centrifugation. The overall purification was 130-fold based on enzyme activity. The purified complex contained three major and one minor polypeptide components when analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. These were identified by heat treatment, limited proteolysis and peptide mapping as pyruvate dehydrogenase (E1; Mr 92500), acetyltransferases (E2; major component, Mr 76000, and minor component, Mr 77 800), and lipoamide dehydrogenase (E3; Mr 58000). The purified complex had a sedimentation coefficient of 48S and the specific activity for the overall reaction of the complex was 6.5 μmol substrate transformed (mg protein)−1 min−1 at the optimum pH (7.8) and 25 °C. The lesions in four ace mutants lacking overall pyruvate dehydrogenase complex activity were identified after partial purification of the corresponding cell-free extracts. Three strains, designated aceA mutants, lacked pyruvate dehydrogenase activity (E1 component) and one strain, an aceB mutant, lacked the activity of the acetyltransferase (E2 component).