Enzymology/Molecular Biology of Aldehyde Dehydrogenase

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DESCRIPTION (provided by applicant): 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.