Molecular characterization of an aldehyde/alcohol dehydrogenase gene from Clostridium acetobutylicum ATCC 824.
A gene (aad) coding for an aldehyde/alcohol dehydrogenase (AAD) was identified immediately upstream of the previously cloned ctfA (J. W. Cary, D. J. Petersen, E. T. Papoutsakis, and G. N. Bennett, Appl. Environ. Microbiol. 56:1576-1583, 1990) of Clostridium acetobutylicum ATCC 824 and sequenced. The 2,619-bp aad codes for a 96,517-Da protein. Primer extension analysis identified two transcriptional start sites 83 and 243 bp upstream of the aad start codon. The N-terminal section of AAD shows homology to aldehyde dehydrogenases of bacterial, fungal, mammalian, and plant origin, while the C-terminal section shows homology to alcohol dehydrogenases of bacterial (which includes three clostridial alcohol dehydrogenases) and yeast origin. AAD exhibits considerable amino acid homology (56% identity) over its entire sequence to the trifunctional protein encoded by adhE from Escherichia coli. Expression of aad from a plasmid in C. acetobutylicum showed that AAD, which appears as a approximately 96-kDa band in denaturing protein gels, provides elevated activities of NADH-dependent butanol dehydrogenase, NAD-dependent acetaldehyde dehydrogenase and butyraldehyde dehydrogenase, and a small increase in NADH-dependent ethanol dehydrogenase. A 957-bp open reading frame that could potentially encode a 36,704-Da protein was identified upstream of aad.
Nair
R V
RV
Department of Chemical Engineering, Northwestern University, Evanston, Illinois 60208.
Bennett
G N
GN
Papoutsakis
E T
ET
D000426:Alcohol Dehydrogenase / Q000235:genetics*; D000429:Alcohol Oxidoreductases / Q000235:genetics*; D000444:Aldehyde Dehydrogenase / Q000235:genetics*; D000445:Aldehyde Oxidoreductases / Q000235:genetics*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D003013:Clostridium / Q000201:enzymology / Q000235:genetics*; D005285:Fermentation; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D009097:Multienzyme Complexes; D012333:RNA, Messenger; C085655:AAD protein, Clostridium acetobutylicum; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase; D000445:Aldehyde Oxidoreductases; D000444:Aldehyde Dehydrogenase