Studies on the mechanism of the Oriental variant and on the characterization and the function of Saccharomyces cerevisiae aldehyde dehydrogenases
Abstract
Many Orientals were found to possess a variant form of liver mitochondrial ALDH2, in which glutamate 487 was substituted by a lysine. Some of those people were found to be heterozygous when genotyped for ALDH2. Liver tissue from a heterozygous person was found to possess mRNAs for both the glutamate- and lysine-subunits. The two enzyme forms were coexpressed in E. coli and were separated by isoelectric focusing to show that heterotetramers were formed. The purified enzymes were found to have only one K$\rm\sb{m}$ value for NAD and were found to have a low specific activity, just 16-18% of homotetrameric E487 enzyme. There was no pre-steady state burst of NADH formation, a property found with E487 enzyme. The presence of lysine subunit appeared to make glutamate subunit function more like a low active lysine form. S. cerevisiae ALDH is particularly interesting for the cells are able to use ethanol as a sole carbon and energy source. A DNA encoding an ALDH was found while screening S. cerevisiae genomic library. This 2744 bp DNA contained an open reading frame of 501 amino acids. This ALDH1 was found to be located in cytosol and constitutively expressed. The DNA for ALDH1 was recombinantly expressed in E. coli and it was found to be essentially a NADP-dependent enzyme. The K$\rm\sb{m}$ for NADP and acetaldehyde were 40 and 25 $\mu$M, respectively, and the V$\rm\sb{m}$ was 23.6 mol/min/mg. The activity of the enzyme was simulated 2-4 fold by Ca$\sp{2+},$ Mg$\sp{2+}$ and Mn$\sp{2+}$ ions, not by K$\sp+$ ions which was found to activate S. cerevisiae mitochondrial enzymes. Three ALDHs, cytosolic ALDH1, mitochondrial ALDH2 and ALDH5, were disrupted from the genome of TWY397 and the disruptions were verified by nutrient requirement, PCR, Western blot and IEF. The growth of cytosolic ALDH1 and mitochondrial ALDH2 disrupted strains on ethanol was lower than that of parent strain, suggesting that ALDH1 and ALDH2 might be involved in ethanol metabolism. The ALDH1/ALDH2 double disrupted strain grew slowly even on glucose, indicating that both ALDHs might catalyze the oxidation of acetaldehyde produced in the fermentation.
Degree
Ph.D.
Advisors
Weiner, Purdue University.
Subject Area
Biochemistry|Molecular biology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.