Further studies on enzymes catalyzing allylic rearrangments are proposed. Beta-Hydroxydecanoyl thiol ester dehydrase (E. coli), which equilibrates thiol esters of (R)-3hydroxydecanoic acid, E-2-decenoic acid, and Z-3-decenoic acid, is the key enzyme in the biosynthesis of bacterial unsaturated fatty acids. Dehydrase is rapidly inactivated by the mechanism-based inactivator 3decynoyl-NAC (NAC =N-acetylcysteamine thiol ester), via dehydrase-catalyzed isomerization to 2,3decadienoyl-NAC. The amino acid sequence of dehydrase has recently been determined, and the residue that is modified by 3-decynoyl-NAC (and is therefore implicated as the active site base) was shown to be His-70. The long-term objective is to further knowledge of enzymw structure and function. Such knowledge is critical for the rational design of drup that act at the enzyme level.
The specific aims of this proposal include (a) determination (by 15N NMR) of which of dehydrase's imidazole nitrogens is modified by 3-decynoyl-NAC; (b) synthesis and application of novel enzyme inactivators, with analysis of protein by peptide mapping and multinuclear NMR, in order to identify catalytically important residues other than His-70; (c) synthesis and evaluation of ketone analogs of dehydrase's thiol ester substrates as potential nonhydrolyzable substrates and inhibitors; (d) determinnation of the three-dimensional crystal structures of dehydrase that has been inactivated with novel 3-decynoic acid thiol esters, including the ACP thiol ester; (e) complete characterization of dehydrase by multinuclear NMR, allowing comparison of crystal- and solution-state samples and enabling the identification of dehydrase-ACP contact points; (f) generation of mutant dehydrases, with modified active site residues and modified acyl chain binding regions, to test the roles of amino acid residues that are suspected to be important in catalysis and to generate E. coli strains with altered unsaturated fatty acids; (g) reevaluation of the stereochemical course of the allylic rearrangement catalyzed by the Betahydroxydodecanoyl thiol ester dehydrase component of Brevibacterium ammoniagenes fatty acid synthetase (by 2H-decoupled 1H,13C chemical shift correlation spectroscopy), in order to test for mechanistic unifomity among enzymes catalyzing allylic rearrangements.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM036286-06
Application #
3289952
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Project Start
1985-08-01
Project End
1994-11-30
Budget Start
1991-12-01
Budget End
1992-11-30
Support Year
6
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Purdue University
Department
Type
Schools of Pharmacy
DUNS #
072051394
City
West Lafayette
State
IN
Country
United States
Zip Code
47907