Gene-protein relationships of the alpha-keto acid dehydrogenase complexes of Escherichia coli K12: isolation and characterization of lipoamide dehydrogenase mutants.

Abstract

SUMMARY: Ten independent lipoamide dehydrogenase mutants (lpd) of Escherichia coli were isolated by selecting strains which required supplements of acetate plus succinate for best growth on glucose. They would not grow on unsupplemented medium (except anaerobically) nor would they grow with single supplements of acetate or lipoate, but they responded slowly to lysine plus methionine or succinate. Bacteria-free extracts of the mutants had between 1 and 10% of parental lipoamide dehydrogenase activity and no activity for the pyruvate and α-ketoglutarate dehydrogenase complexes could be detected. Evidence that the mutants contained the dehydrogenase (E1) and transacylase (E2) components of the complexes and were deficient only in the lipoamide dehydrogenase (E3) components was obtained from studies with mixtures containing lpd mutant extracts and either extracts of other mutants having defined lesions or purified lipoamide dehydrogenases, e.g. overall pyruvate dehydrogenase complex could be reconstituted with extracts of aceE and F mutants and the α-ketoglutarate complex was similarly reconstituted with sucA and B extracts. Furthermore, both complexes could be restored by adding extract of an aceE, sucA double-amber mutant (which lacks both types of E1 and E2 component but has 30% of parental lipoamide dehydrogenase activity) or with purified bacterial and mammalian lipoamide dehydrogenases. The bacterial enzymes were several times more efficient than the mammalian enzyme for restoring pyruvate dehydrogenase complex activity. Genetic studies indicated that the wild-type phenotype could be restored by single reversion or transduction events and they confirmed that the mutants are deficient only in lipoamide dehydrogenase. The mutant phenotype was introduced into a recipient strain by cotransduction with leu +. This indicates that there is a lipoamide dehydrogenase gene in the leu region of the Escherichia coli linkage map and strongly supports the view that the E3 components of both α-ketoacid dehydrogenase complexes are specified by a single lipoamide dehydrogenase gene (lpd).