Biochemical and genetic characterization of the acetaldehyde dehydrogenase complex from Acetobacter europaeus.
The aldehyde dehydrogenase complex, which catalyzes the oxidation of acetaldehyde to acetic acid, was purified to apparent homogeneity from the membrane fraction of the industrial vinegar-producing strain Acetobacter europaeus. The determined Km for acetaldehyde was 2.1 mM. SDS-PAGE of the enzyme complex showed the presence of three different subunits with molecular masses of 79, 46, and 17 kDa, respectively. The two larger subunits contained heme. The difference spectrum indicated a cytochrome c, a heme B, and a [2Fe-2S] cluster. The nucleotide sequence 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 of 84.1, 49.0, and 16.7 kDa; these were assigned to the purified proteins and named aldH, aldF, and aldG, respectively. The N-terminal sequence of the 79-kDa subunit was detected within the predicted amino acid sequence of AldH, which indicated the presence of a leader peptide. Cotranscription 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 dehydrogenase 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 similarities to the C-terminal part of several pyrroloquinoline-chinone-dependent glucose dehydrogenases.
Thurner
C
C
Institut für Lebensmittelwissenschaft, Labor für Lebensmittelmikrobiologie, Eidgenössische Technische Hochschule, ETH-Zentrum, CH-8092 Zürich, Switzerland.
Vela
C
C
Thöny-Meyer
L
L
Meile
L
L
Teuber
M
M
D000091:Acetobacter / Q000201:enzymology / Q000235:genetics*; D000445:Aldehyde Oxidoreductases / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003067:Coenzymes; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D006418:Heme / Q000031:analogs & derivatives; D007506:Iron-Sulfur Proteins / Q000235:genetics; D008667:Metalloproteins; D008969:Molecular Sequence Data; D000090006:Molybdenum Cofactors; D009097:Multienzyme Complexes / Q000235:genetics*; D005810:Multigene Family; D011621:Pteridines; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
D000445:Aldehyde Oxidoreductases; D003067:Coenzymes; D004269:DNA, Bacterial; D006418:Heme; D007506:Iron-Sulfur Proteins; D008667:Metalloproteins; D000090006:Molybdenum Cofactors; D009097:Multienzyme Complexes; D011621:Pteridines; C031690:heme C; C022261:molybdopterin; C018699:aldehyde dehydrogenase (NAD(P)+)