Molecular biological studies on liver mitochondrial aldehyde dehydrogenase

The tools of molecular biology were applied to study different facets of liver aldehyde dehydrogenase. One point investigated was the sequence of the various liver forms of the enzyme and the structure of the genes coding for the enzyme. It was found that an intron existed between the gene sequence coding for amino acids 21 and 22. The presence of the intron could explain why no sequence identity exists between the first 21 residues of the cytosol and mitochondrial isozymes while there is 70% identity between the remaining amino acids. Having the cDNA coding for the mitochondrial enzyme allowed us to produce the precursor form of the enzyme in rabbit reticulocytes and perform in vitro import into isolated mitochondria where it was found that alcohols caused an inhibition of import. The active enzyme was expressed in E. coli and site directed mutagenesis was performed to probe for the active site residues. The role of cysteine and position 302 and glutamate at position 487 was studied.