BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF THE ALCALIGENES-EUTROPHUS PYRUVATE-DEHYDROGENASE COMPLEX AND IDENTIFICATION OF A NEW-TYPE OF DIHYDROLIPOAMIDE DEHYDROGENASE

Sequence analysis of a 6.3-kbp genomic EcoRI-fragment of Alcaligenes e utrophus, which was recently identified by using a dihydrolipoamide de hydrogenase-specific DNA probe (A. Pries, S. Hein, and A. Steinbuchel, FEMS Microbiol. Lett. 97:227-234, 1992), and of an adjacent 1.0-kbp E coRI fragment revealed the structural genes of the A. eutrophus pyruva te dehydrogenase complex, pdhA (2,685 bp), pdhB (1,659 bp), and pdhL ( 1,782 bp), encoding the pyruvate dehydrogenase (El), the dihydrolipoam ide acetyltransferase (E2), and the dihydrolipoamide dehydrogenase (E3 ) components, respectively. Together with a 675-bp open reading frame (ORF3), the function of which remained unknown, these genes occur coli nearly in one gene cluster in the order pdhA, pdhB, ORF3, and pdhL. Th e A. eutrophus pdhA, pdhB, and pdhL gene products exhibited significan t homologies to the El, E2, and E3 components, respectively, of the py ruvate dehydrogenase complexes of Escherichia coli and other organisms . Heterologous expression of pdhA, pdhB, and pdhL in E. coli K38(pGP1- 2) and in the aceEF deletion mutant E. coli YYC202 was demonstrated by the occurrence of radiolabeled proteins in electropherograms, by spec trometric detection of enzyme activities, and by phenotypic complement ation, respectively. A three-step procedure using chromatography on DE AE-Sephacel, chromatography on the triazine dye affinity medium Procio n Blue H-ERD, and heat precipitation purified the E3 component of the A. eutrophus pyruvate dehydrogenase complex from the recombinant E. co li K38(pGP1-2, pT7-4SH7.3) 60-fold, recovering 41.5% of dihydrolipoami de dehydrogenase activity. Microsequencing of the purified E3 componen t revealed an amino acid sequence which corresponded to the N-terminal amino acid sequence deduced from the nucleotide sequence of pdhL. The N-terminal region of PdhL comprising amino acids 1 to 112 was disting uished from all other known dihydrolipoamide dehydrogenases. It resemb led the N terminus of dihydrolipoamide acyltransferases, and it contai ned one single lipoyl domain which was separated by an adjacent hinge region from the C-terminal region of the protein that exhibited high h omology to classical dihydrolipoamide dehydrogenases.