MOLECULAR-CLONING, CHARACTERIZATION, AND POTENTIAL ROLES OF CYTOSOLICAND MITOCHONDRIAL ALDEHYDE DEHYDROGENASES IN ETHANOL-METABOLISM IN SACCHAROMYCES-CEREVISIAE

The full-length DNAs for two Saccharomyces cerevisiae aldehyde dehydro genase (ALDH) genes were cloned and expressed in Escherichia coli, A 2 ,744-bp DNA fragment contained an open reading frame encoding cytosoli c ALDH1, with 500 amino acids, which was located on chromosome XVI. A 2,661-bp DNA fragment contained an open reading frame encoding mitocho ndrial ALDH5, with 519 amino acids, of which the N-terminal 23 amino a cids were identified as the putative leader sequence, The ALDH5 gene w as located on chromosome V. The commercial ALDH (designated ALDH2) was partially sequenced and appears to be a mitochondrial enzyme encoded by a gene located on chromosome XV. The recombinant ALDH1 enzyme was f ound to be essentially NADP dependent, while the ALDH5 enzyme could ut ilize either NADP or NAD as a cofactor, The activity of ALDH1 was stim ulated two- to fourfold by divalent cations but was unaffected by K+ i ons. In contrast, the activity of ALDH5 increased in the presence of K + ions: Ii-fold with NADP and 40-fold with NAD, respectively, Activity staining of isoelectric focusing gels shelved that cytosolic ALDH1 co ntributed 30 to 70% of the overall activity, depending on the cofactor used, while mitochondrial ALDH2 contributed the rest, Neither ALDH5 n or the other ALDH-like proteins identified from the genomic sequence c ontributed to the in vitro oxidation of acetaldehyde. To evaluate the physiological roles of these three ALDH isoenzymes, the genes encoding cytosolic ALDH1 and mitochondrial ALDH2 and ALDH5 were disrupted in t he genome of strain TWY397 separately or simultaneously. The growth of single-disruption Delta ald1 and Delta ald2 strains on ethanol was ma rginally slower than that of the parent strain, The Delta ald1 Delta a ld2 double-disruption strain failed to grow on glucose alone, but grow th was restored by the addition of acetate, indicating that both ALDHs might catalyze the oxidation of acetaldehyde produced during fermenta tion, The double-disruption strain grew very slowly on ethanol, The ro le of mitochondrial ALDH5 in acetaldehyde metabolism has not been defi ned but appears to be unimportant.