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.

Public Health Relevance

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.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
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Special Emphasis Panel (ZRG1)
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Ernst, Nancy L
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University of Illinois Urbana-Champaign
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Cronan, John E (2018) Advances in synthesis of biotin and assembly of lipoic acid. Curr Opin Chem Biol 47:60-66
Manandhar, Miglena; Cronan, John E (2018) A Canonical Biotin Synthesis Enzyme, 8-Amino-7-Oxononanoate Synthase (BioF), Utilizes Different Acyl Chain Donors in Bacillus subtilis and Escherichia coli. Appl Environ Microbiol 84:
Cao, Xinyun; Zhu, Lei; Song, Xuejiao et al. (2018) Protein moonlighting elucidates the essential human pathway catalyzing lipoic acid assembly on its cognate enzymes. Proc Natl Acad Sci U S A 115:E7063-E7072
Srinivas, Swaminath; Cronan, John E (2017) An Eight-Residue Deletion in Escherichia coli FabG Causes Temperature-Sensitive Growth and Lipid Synthesis Plus Resistance to the Calmodulin Inhibitor Trifluoperazine. J Bacteriol 199:
Manandhar, Miglena; Cronan, John E (2017) Pimelic acid, the first precursor of the Bacillus subtilis biotin synthesis pathway, exists as the free acid and is assembled by fatty acid synthesis. Mol Microbiol 104:595-607
Cao, Xinyun; Zhu, Lei; Hu, Zhe et al. (2017) Expression and Activity of the BioH Esterase of Biotin Synthesis is Independent of Genome Context. Sci Rep 7:2141
Bi, Hongkai; Zhu, Lei; Jia, Jia et al. (2016) A Biotin Biosynthesis Gene Restricted to Helicobacter. Sci Rep 6:21162
Henke, Sarah K; Cronan, John E (2016) The Staphylococcus aureus group II biotin protein ligase BirA is an effective regulator of biotin operon transcription and requires the DNA binding domain for full enzymatic activity. Mol Microbiol 102:417-429
Cronan, John E (2016) pBR322 vectors having tetracycline-dependent replication. Plasmid 84-85:20-6
Bi, Hongkai; Zhu, Lei; Jia, Jia et al. (2016) Unsaturated Fatty Acid Synthesis in the Gastric Pathogen Helicobacter pylori Proceeds via a Backtracking Mechanism. Cell Chem Biol 23:1480-1489

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