There are 17 genes coding for various forms of human aldehyde dehydrogenase (ALDH). The physiological role of most members of the ALDH family is not known though much is known about the structure and mechanisms of a few of the isozymes. An error prone PCR mutational approach has allowed us to produce mutants of some of the isozymes that have properties that differ from those of the parent enzyme. We will build upon this finding to use the mutational approach to select for isozymes that have increased activity as well as enhanced stability as compared to the parent enzyme. The rate-limiting step is the class 1, 2 and 3 isozymes, enzymes whose structure and mechanism have been studied, are different in each enzyme form. We will introduce the mutations found with any one isozyme into the others to determine if we can increase the same step in the catalytic process. The purpose is both to learn more about what controls the rate limiting step in each isozyme and then try to improve the catalytic efficiencies in the other isozymes using information gain from a different isozyme. Isozymes with increased stability will be identified from the screening procedure and then studied both in vitro and in vivo with the goal of producing a more stable enzyme. In addition we will try to select for an enzyme with an enhanced ability to oxidize aldophophamide, a toxic aldehyde produced during the transformation of the pro-drug cyclophosphamide, a compound that will destroy some tumors. Unfortunately, the drug will kill any cell that does not have an ALDH to protect it by oxidizing the toxic aldehyde. Bone marrow is one such cell. If we can produce a mutant with enhanced activity against aldophophamide we will use cell culture work to determine if cells transformed with the more active mutant ALDH can protect the cell from the toxic effects of the compound. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Research Project (R01)
Project #
5R01AA005812-20
Application #
6905680
Study Section
Alcohol and Toxicology Subcommittee 4 (ALTX)
Program Officer
Velazquez, Jose M
Project Start
1983-06-15
Project End
2008-05-31
Budget Start
2005-06-01
Budget End
2006-05-31
Support Year
20
Fiscal Year
2005
Total Cost
$350,955
Indirect Cost
Name
Purdue University
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
072051394
City
West Lafayette
State
IN
Country
United States
Zip Code
47907
Mukhopadhyay, Abhijit; Wei, Baoxian; Weiner, Henry (2013) Mitochondrial NAD dependent aldehyde dehydrogenase either from yeast or human replaces yeast cytoplasmic NADP dependent aldehyde dehydrogenase for the aerobic growth of yeast on ethanol. Biochim Biophys Acta 1830:3391-8
Weiner, Henry; Duester, Greg; Maser, Edmund et al. (2009) Enzymology and molecular biology of carbonyl metabolism. Introduction. Chem Biol Interact 178:1
Ho, Kwok Ki; Mukhopadhyay, Abhijit; Li, Yi Feng et al. (2008) A point mutation produced a class 3 aldehyde dehydrogenase with increased protective ability against the killing effect of cyclophosphamide. Biochem Pharmacol 76:690-6
Mukhopadhyay, Abhijit; Weiner, Henry (2007) Delivery of drugs and macromolecules to mitochondria. Adv Drug Deliv Rev 59:729-38
Mukhopadhyay, Abhijit; Yang, Chun-song; Wei, Baoxian et al. (2007) Precursor protein is readily degraded in mitochondrial matrix space if the leader is not processed by mitochondrial processing peptidase. J Biol Chem 282:37266-75
Brichac, Jiri; Ho, Kwok Ki; Honzatko, Ales et al. (2007) Enantioselective oxidation of trans-4-hydroxy-2-nonenal is aldehyde dehydrogenase isozyme and Mg2+ dependent. Chem Res Toxicol 20:887-95
Rodriguez-Zavala, Jose Salud; Allali-Hassani, Abdellah; Weiner, Henry (2006) Characterization of E. coli tetrameric aldehyde dehydrogenases with atypical properties compared to other aldehyde dehydrogenases. Protein Sci 15:1387-96
Ho, Kwok Ki; Hurley, Thomas D; Weiner, Henry (2006) Selective alteration of the rate-limiting step in cytosolic aldehyde dehydrogenase through random mutagenesis. Biochemistry 45:9445-53
Mukhopadhyay, Abhijit; Zullo, Steven J; Weiner, Henry (2006) Factors that might affect the allotopic replacement of a damaged mitochondrial DNA-encoded protein. Rejuvenation Res 9:182-90
Ho, Kwok Ki; Weiner, Henry (2005) Isolation and characterization of an aldehyde dehydrogenase encoded by the aldB gene of Escherichia coli. J Bacteriol 187:1067-73

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