This work aims at understanding aldehyde dehydrogenase (A1DH) function at the molecular (atomic) level. Liver A1DH acts in human alcohol metabolism, clearing ethanol-derived acetaldehyde, via conversion to acetate. Selective loss of either of the two major liver forms of A1DH (via disulfiram treatment or congenital absence common in Orientals) leads to aversion to alcohol. Chemical modifications and primary structural data from diverse AlDHs have partially defined functional regions. However, lack of three-dimensional data on AlDHs now severely limits further interpretations of enzyme function. Therefore, crystallographic study of the rat Class 3 A1DH is proposed. This A1DH form is chosen for several intrinsic characteristics as well as for the availability of an E. coli system expressing large amounts of the this A1DH in active non-fusion form. In the funding period, emphasis will be on crystallization, derivitization of crystals with heavy atoms, collection of X-ray diffraction data and aiming at solving the tertiary structure of the enzyme. Tertiary structure will also be addressed by chemical modification aimed at crosslinking an internal salt bridge in Class 1/2 AlDHs suggested from alignment of AlDHs with thiol proteases and papain crystal structure correlations. Concurrently, site-directed mutagenesis on this A1DH will also be performed. Specific mutants of Cys-302 and Glu-268 (Class 1 and 2 numbering) will be characterized regarding kinetic properties, for evaluation relative to the tertiary structure as it becomes available. The primary structure of a membrane-bound A1DH from rat liver remains under investigation, via both peptide analyses and cDNA library screenings. This microsomal enzyme structure will be the first of a membrane-bound A1DH, and will provide significant insights regarding structure/function relationships of AlDHs.

National Institute of Health (NIH)
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
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Biochemistry, Physiology and Medicine Subcommittee (ALCB)
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University of Pittsburgh
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