IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF THE ACO GENES ENCODING THE PELOBACTER-CARBINOLICUS ACETOIN DEHYDROGENASE ENZYME-SYSTEM

Use of oligonucleotide probes, which were deduced from the N-terminal sequences of the purified enzyme components, identified the structural genes for the alpha and beta subunits of El (acetoin:2,6-dichlorophen olindophenol oxidoreductase), E2 (dihydrolipoamide acetyltransferase), and E3 (dihydrolipoamide dehydrogenase) of the Pelobacter carbinolicu s acetoin dehydrogenase enzyme system, which were designated acoA, aco B, acoC, and acoL, respectively. The nucleotide sequences of acoA (979 bp), acoB (1,014 bp), acoC (1,353 bp), and acoL (1,413 bp) as well as of acoS (933 bp), which encodes a protein with an M(r) of 34,421 exhi biting 64.7% amino acid identity to the Escherichia coli lipA gene pro duct, were determined. These genes are clustered on a 6.1-kbp region. Heterologous expression of acoA, acoB, acoC, acoL, and acoS in E. coli was demonstrated. The amino acid sequences deduced from acoA, acoB, a coC, and acoL for E1alpha (M(r), 34,854), E1beta (M(r), 36,184), E2 (M (r), 47,281), and E3 (M(r), 49,394) exhibited striking similarities to the amino acid sequences of the components of the Alcaligenes eutroph us acetoin-cleaving system. Homologies of up to 48.7% amino acid ident ity to the primary structures of the enzyme components of various 2-ox o acid dehydrogenase complexes also were found. In addition, the respe ctive genes of the 2-oxo acid dehydrogenase complexes and of the aceto in dehydrogenase enzyme system were organized very similarly, indicati ng a close relationship of the P. carbinolicus acetoin dehydrogenase e nzyme system to 2-oxo acid dehydrogenase complexes.