Cloning, sequencing, and characterization of the gene encoding the smallest subunit of the three-component membrane-bound alcohol dehydrogenase from Acetobacter pasteurianus.
The membrane-bound alcohol dehydrogenase (ADH) of Acetobacter pasteurianus NCI1452 consists of three different subunits, a 78-kDa dehydrogenase subunit, a 48-kDa cytochrome c subunit, and a 20-kDa subunit of unknown function. For elucidation of the function of the smallest subunit, this gene was cloned from this strain by the oligonucleotide-probing method, and its nucleotide sequence was determined. Comparison of the deduced amino acid sequence and the NH2-terminal sequence determined for the purified protein indicated that the smallest subunit contained a typical signal peptide of 28 amino acids, as did the larger two subunits. This gene complemented the ADH activity of a mutant strain which had lost the smallest subunit. Disruption of this gene on the chromosome resulted in loss of ADH activity in Acetobacter aceti, indicating that the smallest subunit was essential for ADH activity. Immunoblot analyses of cell lysates prepared from various ADH mutants suggested that the smallest subunit was concerned with the stability of the 78-kDa subunit and functioned as a molecular coupler of the 78-kDa subunit to the 48-kDa subunit on the cytoplasmic membrane.
Kondo
K
K
Department of Biotechnology, University of Tokyo, Japan.
Beppu
T
T
Horinouchi
S
S
D000091:Acetobacter / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D000426:Alcohol Dehydrogenase / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002451:Cell Compartmentation; D003001:Cloning, Molecular; D000431:Ethanol / Q000378:metabolism*; D005798:Genes, Bacterial / Q000235:genetics*; D005816:Genetic Complementation Test; D008565:Membrane Proteins / Q000235:genetics*; D008566:Membranes / Q000201:enzymology; D008954:Models, Biological; D008969:Molecular Sequence Data; D016296:Mutagenesis; D015345:Oligonucleotide Probes; D010084:Oxidation-Reduction; D011487:Protein Conformation
D008565:Membrane Proteins; D015345:Oligonucleotide Probes; D000431:Ethanol; D000426:Alcohol Dehydrogenase