Molecular Cloning, Characterization, and Potential Roles of Cytosolic and Mitochondrial Aldehyde Dehydrogenases in Ethanol Metabolism in Saccharomyces cerevisiae

Yeast aldehyde dehydrogenase (ALDH) has been studied for many years. In the 1950s it was found that a K+ ion-activated enzyme existed, and many kinetic properties of the enzyme were studied by different investigators (2, 3, 21). Most of the interest was focused on understanding the metabolism of acetaldehyde derived from the oxidation of ethanol during the aerobic growth of the organism. More recently, a different form of ALDH was identified which primarily oxidized long-chain aldehydes and was not involved in acetaldehyde oxidation (1, 25). Our laboratory reported on the cloning and sequencing of a yeast ALDH (19) and suggested it was mitochondrial in origin. When we started to reinvestigate the project, we came to realize that the DNA we sequenced could not have been totally correct. Hence, we rescreened our libraries and found two new DNAs which would code for different enzyme forms. The complete Saccharomyces cerevisiae genome has recently been published (7), and it verified that our original clone was not correct but that the two new genes we had found were present. Here we report data on the cloning, expression, purification, and characterization of these two enzyme forms. Neither is the commercially available K+-activated enzyme. During growth on ethanol it is necessary for S. cerevisiae to convert two carbons from ethanol to acetate so they can be used by the glyoxylate system to incorporate the carbons into usable metabolites. The total genomic sequence of S. cerevisiae S288C revealed that seven genes could code for proteins which appear to be members of the ALDH family. The recombinantly expressed enzymes encoded by the two genes we cloned appeared to have properties similar to enzymes found by other investigators. One enzyme was shown to be localized to the mitochondria, with properties somewhat similar, but not identical, to those reported by Black in 1951 (2). This enzyme was also shown to exhibit properties different from those of the commercially available enzyme. The second enzyme was localized to the cytosol, and had properties very similar to those of the enzyme described by Seegmiller (20) and Dickinson (4). The data also suggest that the acetaldehyde produced during ethanol oxidation could be converted into acetate in the cytosol as well as in the mitochondria. The contribution of the enzymes may be influenced by the NADP/NAD ratio in the cells. Investigators have suggested that mitochondrial ALDH was involved in the oxidative metabolism of ethanol while cytosolic ALDH was responsible for the oxidation of acetaldehyde produced during fermentation (15, 18), although the bulk of acetaldehyde is reduced to ethanol. We have used the nomenclature previously adopted (31) to identify the yeast enzymes described in this study based on their amino acid sequence homologies with and enzymatic similarities to the mammalian enzymes. We have disrupted the cytosolic ALDH gene (designated ALD1) and two mitochondrial ALDH genes (designated ALD2 and ALD5) in the S. cerevisiae genome and found that ALDH1 and ALDH2 were indeed important for the metabolism of ethanol. Both ALDHs might be involved in the oxidation of acetaldehyde formed during fermentation. The physiological role of ALDH5 in acetaldehyde metabolism was not completely elucidated in this study.