Studies on the fate of S-(3-decynoyl)-N-acetylcysteamine in Escherichia coli
Abstract
$\beta$-Hydroxydecanoyl thiol ester dehydrase catalyzes the branching step in the biosynthetic pathway for unsaturated fatty acids in E. coli. This enzyme catalyzes the dehydration of 3-hydroxydecanoyl thiol esters to (Z)-3-decenoyl esters. Dehydrase is irreversibly inhibited by S-(3-decynoyl)-N-acetyl-cysteamine (3-decynoyl-NAC), providing the first example of mechanism-based, or "suicide," enzyme inactivation. The structure of the histidine adduct that is formed during this inactivation is examined in this study. In addition, the role of 3-decynoyl-NAC in fatty acid degradation has also been determined. The fabA gene, which codes for dehydrase, was cloned using template directed DNA amplification, and overexpressed in E. coli. Each histidine residue was changed to asparagine using site-directed mutagenesis. The histidine-129 mutant protein was labeled with $\sp{15}$N and the labeled protein was studied using nuclear magnetic resonance spectroscopy. The expression system developed in this study allows the purification of twenty-five to fifty milligrams of labeled protein from a liter of culture medium. NMR analysis of the mutant protein, which shows identical catalytic activity to the wild type protein, allows assignment of the histidine imidazole $\sp{15}$N resonances. Previous work has shown that "pyridine-like" nitrogens resonate downfield of "pyrrole-like" nitrogens. In labeled dehydrase, N$\sp{\varepsilon 2}$ resonates downfield relative to N$\sp{\delta 1}$, indicating that the imidazole is in the N$\sp{\delta 1}$--H tautomer. Upon inactivation, the signals are reversed, indicating that the imidazole of histidine-70 becomes alkylated on N$\sp{\varepsilon 2}$. These results implicate N$\sp{\varepsilon 2}$ as the locus of active site basicity. The imidazole $\sp{15}$N chemical shifts suggest that N$\sp{\varepsilon 1}$ is involved in a hydrogen bond, while N$\sp{\varepsilon 2}$ is not. This interaction should stabilize the observed tautomeric form of the imidazole. The mechanistic implications of this hydrogen bond are discussed. The ability of the fatty acid oxidation complex of E. coli to isomerize 3-decynoyl-NAC to 2,3-decadienoyl-NAC was also determined. Studies of the isomerase and oxidation activities show that the two activities co-purify. Studies of the levels of the enzyme activities in crude homogenates of mutants suggest that the isomerase activity is catalyzed by the fatty acid oxidation complex.
Degree
Ph.D.
Advisors
Schwab, Purdue University.
Subject Area
Biochemistry|Biophysics|Molecular biology
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