Characterization of the genes encoding the three-component membrane-bound alcohol dehydrogenase from Gluconobacter suboxydans and their expression in Acetobacter pasteurianus.
The three-component membrane-bound alcohol dehydrogenase (ADH) of Gluconobacter suboxydans IFO12528 was purified, and the NH2-terminal amino acid sequence of each subunit was determined. On the basis of the amino acid sequences, the genes adhA, encoding the 72-kDa dehydrogenase, adhB, encoding the 44-kDa cytochrome c-553 (a CO-binding cytochrome c), and adhS, encoding a 15-kDa protein, were cloned and the amino acid sequences of their products were deduced from the nucleotide sequences. The dehydrogenase and cytochrome genes were clustered with the same transcription polarity, as is the case in species of Acetobacter, another genus of acetic acid bacteria. These AdhA and AdhB subunits showed similarity in amino acid sequence to those from Acetobacter spp., whereas AdhS showed no similarity to the corresponding subunit of the ADH complex of Acetobacter pasteurianus. Consistent with this, adhS of G. suboxydans could not complement a defect in the corresponding subunit of A. pasteurianus. When the adhA-adhB gene cluster of G. suboxydans was expressed in an ADH-deficient mutant of A. pasteurianus, the transformant showed distinct ADH activity. The ADH complex was purified to near homogeneity and consisted of two subunits, the dehydrogenase and the cytochrome c subunits derived from G. suboxydans, without any other subunit. These data suggested that AdhS, the smallest subunit of ADH, from G. suboxydans is not essential for ADH activity in A. pasteurianus, in contrast to the essential role of A. pasteurianus AdhS, which is required for correct assembly of the dehydrogenase and cytochrome c subunits on the membrane.
Kondo
K
K
Department of Biotechnology, Graduate School of Agriculture and Life Sciences, University of Tokyo, Japan.
Horinouchi
S
S
D000091:Acetobacter / Q000235:genetics*; D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D005810:Multigene Family; D016366:Open Reading Frames; D011548:Pseudomonadaceae / Q000201:enzymology* / Q000235:genetics
D000426:Alcohol Dehydrogenase