ALCALIGENES-EUTROPHUS POSSESSES A 2ND PYRUVATE-DEHYDROGENASE (E1)

Two gene loci, which hybridized with pdhA, the structural gene of the E1 component of the Alcaligenes eutrophus pyruvate dehydrogenase compl ex [Hein, S. & Steinbuchel, A. (1994) J. Bacteriol. 176, 4394-4408], w ere identified on two nonrelated A. eutrophus chromosomal BamHI fragme nts by using a pdhA-specific DNA probe. These data indicated that A. e utrophus possesses, beside PdhA, two additional distinct pyruvate dehy drogenases (E1). A 6.8-kbp genomic BamHI fragment of A. eutrophus was cloned, and sequence analysis of a 3.896-kbp region revealed the struc tural gene pdhE (2.694 kbp) for a second pyruvate dehydrogenase (El), which was not clustered with structural genes for other components of 2-oxo acid dehydrogenase complexes. The A. eutrophus pdhE gene product (898 amino acid residues) exhibited significant similarities to the E l components of the pyruvate dehydrogenase complexes of A. eutrophus, Neisseria meningitidis, Escherichia coli and Azotobacter vinelandii, w hich are also composed of only one type of subunit. Heterologous expre ssion of pdhE in the aceEF deletion mutant E. coli YYC202 was demonstr ated by spectrometric detection of enzyme activities and by phenotypic complementation to acetate prototrophy. These complementation studies indicated that the El component of the A. eutrophus pyruvate dehydrog enase complex can be replaced by a functionally active pdhE gene produ ct.