ABSTRACT
Four additional variants of alcohol and aldehyde dehydrogenases have been purified and functionally characterized, and their primary structures have been determined. The results allow conclusions about the structural and evolutionary relationships within the large family of MDR alcohol dehydrogenases from characterizations of the pigeon (Columba livia) and dogfish (Scyliorhinus canicula) major liver alcohol dehydrogenases. The pigeon enzyme turns out to be of class I type and the dogfish enzyme of class III type. This result gives a third type of evidence, based on purifications and enzyme characterization in lower vertebrates, that the classical liver alcohol dehydrogenase originated by a gene duplication early in the evolution of vertebrates. It is discernable as the major liver form at about the level in-between cartilaginous and osseous fish. The results also show early divergence within the avian orders. Structures were determined by Edman degradations, making it appropriate to acknowledge the methodological contributions of Pehr Edman during the 65 years since his thesis at Karolinska Institutet, where also the present analyses were performed.
Subject(s)
Alcohol Dehydrogenase/chemistry , Alcohol Dehydrogenase/genetics , Aldehyde Oxidoreductases/chemistry , Aldehyde Oxidoreductases/genetics , Gene Duplication , Genes, Duplicate , Alcohol Dehydrogenase/isolation & purification , Alcohol Dehydrogenase/metabolism , Aldehyde Oxidoreductases/isolation & purification , Aldehyde Oxidoreductases/metabolism , Amino Acid Sequence , Animals , Columbidae , Cricetinae , Dogfish , Evolution, Molecular , Liver/enzymology , Molecular Sequence Data , PhylogenyABSTRACT
Mushroom alcohol dehydrogenase (ADH) from Agaricus bisporus (common mushroom, champignon) was purified to apparent homogeneity. One set of ADH isozymes was found, with specificity against formaldehyde/glutathione. It had two highly similar subunits arranged in a three-member isozyme set of dimers with indistinguishable activity. Determination of the primary structure by a combination of chemical, mass spectrometric and cDNA sequence analyses, correlated with molecular modeling towards human ADHs, showed that the active site residues are of class III ADH type, and that the subunit differences affect other residues. Class I and III forms of ADHs characterized define conserved substrate-binding residues (three and eight, respectively) useful for recognition of these enzymes in any organism.
Subject(s)
Agaricus/enzymology , Aldehyde Oxidoreductases/chemistry , Aldehyde Oxidoreductases/isolation & purification , Base Sequence , Binding Sites , Evolution, Molecular , Fungal Proteins/chemistry , Fungal Proteins/isolation & purification , Isoenzymes/chemistry , Isoenzymes/isolation & purification , Models, Molecular , Molecular Sequence Data , Protein Subunits , Sequence Analysis, DNA , Structural Homology, ProteinABSTRACT
Ethanol oxidation by nicotinoprotein alcohol dehydrogenase (np-ADH) from the bacterium Amycolatopsis methanolica is inhibited by trans-4-(N,N-dimethylamino)-cinnamaldehyde through direct binding to the catalytic zinc ion in a substrate-like geometry. This binding is accompanied by a characteristic red shift of the aldehyde absorbance from 398 nm to 467 nm. Np-ADH is structurally related to mammalian ADH class I, and a model of np-ADH shows how the cinnamaldehyde derivative can be accommodated in the active site of the nicotinoprotein, correlating the structural and enzymological data.
