Fatty acids are responsible for the hydrophobic barrier properties of biological membranes. Barrier quality depends on the species of fatty acid present in the complex lipids (generally phospholipids). In the case of bacteria the rate of fatty acid synthesis must match the growth rate of the cells. We propose to study the regulation of the subunit composition of acetyl-CoA carboxylase, the complex enzyme responsible for synthesis of malonyl-CoA, the building block of fatty acid synthesis. Our prior work has shown that acetyl-CoA carboxylase is a rate limiting step in Escherichia coli fatty acid synthesis, but it remains a mystery how the stoichiometry of the four subunits of the enzyme is determined. A difficulty in approaching this problem is that all four acetyl-CoA carboxylase genes are essential for growth. We propose a method to bypass the essentiality to allow the stoichiometry and growth rate control of acetyl-CoA carboxylase assembly is regulated. In addition to providing membrane fatty acid moieties, fatty acid synthesis is required for synthesis of two key coenzymes, lipoic acid and biotin, both of which must be covalently attached to their cognate enzyme proteins to function. Lipoic acid is a key cofactor for both aerobic and single carbon metabolism. In our prior work we discovered the first lipoic acid synthesis pathway, that of E. coli, and found that the cofactor is assembled on its cognate proteins, rather than first being assembled and then attached. Assembly on site was also seen in a more complex pathway, that of Bacillus subtilis, that required four proteins rather than the two proteins required by E. coli. The phenotypes of B. subtilis strains blocked in lipoic acid assembly are strikingly similar to those of human patients unable to assemble lipoylated proteins. Indeed, these similarities strongly suggest that the pathways put forth by laboratories investigating these disorders are incorrect. We believe that one of the human proteins has an enzyme activity that differs from that usually ascribed and have preliminary data to support this hypothesis. We propose to determine the pathway of lipoate synthesis in humans. Biotin is required throughout biology but is only synthesized by bacteria, archaea, fungi and plants. Although the mechanisms of the highly conserved enzymes responsible for assembling the fused heterocyclic rings of biotin were worked out years ago, the metabolic source of the biotin valeric acid ?tail? that contributes most of the biotin carbons atoms was unknown. Although the carbons were known to be derived from pimelic acid, a ?, ?-dicarboxylic acid, the mechanism of pimelic acid synthesis was unknown in any organism. We showed that in E. coli the pimelate moiety is made deceiving the fatty acid synthesis pathway into making a dicarboxylic acid rather than the usual monocarboxylic acids. This pathway seems to explain pimelate synthesis in most bacteria, but two group of bacteria use different pathways. These are B. subtilis and its close relatives, but not other bacilli and the ?-proteobacteria. We propose to determine these pathways.
Fatty acid synthesis is essential for both membrane lipid bilayer function as well as for synthesis of two key metabolic cofactors, biotin and lipoic acid. This proposal addresses the regulation of the fatty acid synthesis pathway and its role in synthesis of biotin and lipoic acid in diverse organisms.
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