BIOCHEMICAL AND GENETIC-CHARACTERIZATION OF THE ACETALDEHYDE DEHYDROGENASE COMPLEX FROM ACETOBACTER-EUROPAEUS

The aldehyde dehydrogenase complex, which catalyzes the oxidation of a cetaldehyde to acetic acid, was purified to apparent homogeneity from the membrane fraction of the industrial vinegar-producing strain Aceto bacter europaeus. The determined K-m for acetaldehyde was 2.1 mM. SDS- PAGE of the enzyme complex showed the presence of three different subu nits with molecular masses of 79, 46, and 17 kDa, respectively. The tw o larger subunits contained heme. The difference spectrum indicated a cytochrome c, a heme B, and a [2Fe-2S] cluster. The nucleotide sequenc e of several cloned fragments of a 6-kb chromosomal DNA segment from A . europaeus was determined. It contains three consecutive open reading frames that correspond to proteins with calculated molecular masses o f 84.1, 49.0, and 16.7 kDa; these were assigned to the purified protei ns and named aldH, aldF, and aldG, respectively. The N-terminal sequen ce of the 79 kDa subunit was detected within the predicted amino acid sequence of AldH, which indicated the presence of a reader peptide. Co transcription of the three genes was shown by Northern hybridization. Sequence analysis and experimental evidence allowed the assignment of the following cofactors to the respective subunits of the aldehyde deh ydrogenase complex: heme C to AldF, [2Fe-2S] cluster to AldG, and heme B and a molybdopterin cofactor to AldH. Part of an open reading frame , gdhA, was detected upstream of the operon that showed high similarit ies to the C-terminal part of several pyrroloquinoline-chinone-depende nt glucose dehydrogenases.