Dataset Information

Cloning, sequencing, and characterization of the gene encoding the smallest subunit of the three-component membrane-bound alcohol dehydrogenase from Acetobacter pasteurianus.

ABSTRACT: 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.

SUBMITTER: Kondo K

PROVIDER: S-EPMC177283 | biostudies-other | 1995 Sep

REPOSITORIES: biostudies-other

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Cloning, sequencing, and characterization of the gene encoding the smallest subunit of the three-component membrane-bound alcohol dehydrogenase from Acetobacter pasteurianus.

Kondo K K Beppu T T Horinouchi S S

Journal of bacteriology 19950901 17

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 p ...[more]

PMID: 7665483

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Project description:BackgroundCommon beans (Phaseolus vulgaris L.) are widely consumed as a source of proteins and natural products. However, its yield needs to be increased. In line with the agenda of Phaseomics (an international consortium), work of expressed sequence tags (ESTs) generation from bean pods was initiated. Altogether, 5972 ESTs have been isolated. Alcohol dehydrogenase (AD) encoding gene cDNA was a noticeable transcript among the generated ESTs. This AD is an important enzyme; therefore, to understand more about it this study was undertaken.ObjectiveThe objective of this study was to elucidate P. vulgaris L. AD (PvAD) gene cDNA sequence and to predict the three-dimensional (3D) structure of deduced protein.Materials and methodspositive and negative strands of the PvAD cDNA clone were sequenced using M13 forward and M13 reverse primers to elucidate the nucleotide sequence. Deduced PvAD cDNA and protein sequence was analyzed for their basic features using online bioinformatics tools. Sequence comparison was carried out using bl2seq program, and tree-view program was used to construct a phylogenetic tree. The secondary structures and 3D structure of PvAD protein were predicted by using the PHYRE automatic fold recognition server.ResultsThe sequencing results analysis showed that PvAD cDNA is 1294 bp in length. It's open reading frame encodes for a protein that contains 371 amino acids. Deduced protein sequence analysis showed the presence of putative substrate binding, catalytic Zn binding, and NAD binding sites. Results indicate that the predicted 3D structure of PvAD protein is analogous to the experimentally determined crystal structure of s-nitrosoglutathione reductase from an Arabidopsis species.ConclusionsThe 1294 bp long PvAD cDNA encodes for 371 amino acid long protein that contains conserved domains required for biological functions of AD. The predicted deduced PvAD protein's 3D structure reflects the analogy with the crystal structure of Arabidopsis thaliana s-nitrosoglutathione reductase. Further study is required to validate the predicted structure.

Project description:BACKGROUND: The Abeta-binding alcohol dehydrogenase/17beta-hydroxysteroid dehydrogenase type 10 (ABAD/HSD10) is an enzyme involved in pivotal metabolic processes and in the mitochondrial dysfunction seen in the Alzheimer's disease. Here we use comparative genomic analyses to study the evolution of the HADH2 gene encoding ABAD/HSD10 across several eukaryotic species. RESULTS: Both vertebrate and nematode HADH2 genes showed a six-exon/five-intron organization while those of the insects had a reduced and varied number of exons (two to three). Eutherian mammal HADH2 genes revealed some highly conserved noncoding regions, which may indicate the presence of functional elements, namely in the upstream region about 1 kb of the transcription start site and in the first part of intron 1. These regions were also conserved between Tetraodon and Fugu fishes. We identified a conserved alternative splicing event between human and dog, which have a nine amino acid deletion, causing the removal of the strand betaF. This strand is one of the seven strands that compose the core beta-sheet of the Rossman fold dinucleotide-binding motif characteristic of the short chain dehydrogenase/reductase (SDR) family members. However, the fact that the substrate binding cleft residues are retained and the existence of a shared variant between human and dog suggest that it might be functional. Molecular adaptation analyses across eutherian mammal orthologues revealed the existence of sites under positive selection, some of which being localized in the substrate-binding cleft and in the insertion 1 region on loop D (an important region for the Abeta-binding to the enzyme). Interestingly, a higher than expected number of nonsynonymous substitutions were observed between human/chimpanzee and orangutan, with six out of the seven amino acid replacements being under molecular adaptation (including three in loop D and one in the substrate binding loop). CONCLUSION: Our study revealed that HADH2 genes maintained a reasonable conserved organization across a large evolutionary distance. The conserved noncoding regions identified among mammals and between pufferfishes, the evidence of an alternative splicing variant conserved between human and dog, and the detection of positive selection across eutherian mammals, may be of importance for further research on ABAD/HSD10 function and its implication in the Alzheimer's disease.

Dataset Information

Cloning, sequencing, and characterization of the gene encoding the smallest subunit of the three-component membrane-bound alcohol dehydrogenase from Acetobacter pasteurianus.

Publications

Cloning, sequencing, and characterization of the gene encoding the smallest subunit of the three-component membrane-bound alcohol dehydrogenase from Acetobacter pasteurianus.

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