Towards understanding physiological function of mitochondrial fatty acid synthesis

Mitochondria are important eukaryotic organelles because they generate adenosine triphosphate (ATP) that is an energy source of the cell. Most of cellular fatty acids are produced in cytosol, but fatty acids can synthesized also in mitochondria. Mitochondrial fatty acid synthesis (mtFAS) is the rather recently discovered pathway, which is conserved in all eukaryotes. The mtFAS occurs via thioester chemistry, where acyl carrier protein (ACP) acts as an acyl group carrier. Human mitochondrial 2E-enoyl thioester reductase (MECR/Etr1) is an mtFAS enzyme responsible for the final reductive step of the pathway. Octanoic acid is one of the products of the mtFAS and is used for endogenous lipoic acid synthesis. Lipoic acid is an essential co-factor for multiple mitochondrial enzyme complexes. Our understanding of the mechanism of protein lipoylation in eukaryotes has improved only recently; we still lack more detailed insights into this essential post-translational modification pathway. Apart from the role of protein lipoylation by producing octanoic acid/ lipoic acid, postulated physiological functions of other fatty acids synthesized by mtFAS are still under investigation.

One aim of this study was to explore the protein lipoylation pathway. Towards this end, we employed various mtFAS and lipoylation deficient yeast strains and developed novel tools to dissect individual steps of protein lipoylation. We propose a new enzymatic function for mitochondrial lipoyl transferase and demonstrate that the human lipoyl transferase LIPT1 can functionally replace the yeast homolog, responding to external lipoic acid supplementation in the presence of an octanoic acid/ lipoic acid activating enzyme. We propose that our experimental setup in yeast offers a reliable and straightforward platform to examine human enzymes and various substrates they can accept in vivo for protein lipoylation. Appropriately modified, it may also be turned into a tool for the discovery of compounds with the potential to serve as medication for patients with certain protein lipoylation deficits.

Another goal was to identify novel functions of longer chain fatty acids produced by the mtFAS using an engineered MECR mutant enzyme as a tool. To identify the functions of synthesized long chain fatty acids, MECR was mutated in such a way that it can’t synthesize fatty longer than octanoic acid anymore. The engineered MECR mutant yeast strain showed respiratory deficiency in vivo, although proteins were still lipoylated. In vitro kinetics analysis indicated that the MECR mutant has an altered substrate preference towards short-chain fatty acids, and the crystal structure revealed the shortened substrate binding cavity. Cellular oxygen consumption measurement of this mutant yeast strain showed a noticeable reduction in mitochondrial respiration. Furthermore, the proteins encoded by mitochondrial DNA were absent, whereas the expression of the nuclear DNA encoded proteins were unchanged in the MECR mutant variant. Surprisingly, droplet digital polymerase chain reaction (ddPCR) studies revealed loss of mitochondrial DNA in this mutant yeast strain that is not previously reported in yeast strains with a mitochondrial enoyl reductase defect, indicating a novel role for mtFAS in mitochondrial DNA maintenance.