Mitochondrial fatty acid synthesis and maintenance of respiratory competent mitochondria in yeast

Mitochondrial FAS (fatty acid synthesis) of type II is a widely conserved process in eukaryotic organisms, with particular importance for respiratory competence and mitochondrial morphology maintenance in Saccharomyces cerevisiae. The recent characterization of three missing enzymes completes the pathway. Etr1p (enoyl thioester reductase) was identified via purification of the protein followed by molecular cloning. To study the link between FAS and cell respiration further, we also created a yeast strain that has FabI enoyl-ACP (acyl-carrier protein) reductase gene from Escherichia coli engineered to carry a mitochondrial targeting sequence in the genome, replacing the endogenous ETR1 gene. This strain is respiratory competent, but unlike the ETR1 wild-type strain, it is sensitive to triclosan on media containing only non-fermentable carbon source. A colony-colour-sectoring screen was applied for cloning of YHR067w/RMD12, the gene encoding mitochondrial 3-hydroxyacyl-ACP dehydratase (Htd2/Yhr067p), the last missing component of the mitochondrial FAS. Finally, Hfa1p was shown to be the mitochondrial acetyl-CoA carboxylase.

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Divergent acyl carrier protein decouples mitochondrial Fe-S cluster biogenesis from fatty acid synthesis in malaria parasites

eLife ◽

10.7554/elife.71636 ◽

2021 ◽

Vol 10 ◽

Author(s):

Seyi Falekun ◽

Jaime Sepulveda ◽

Yasaman Jami-Alahmadi ◽

Hahnbeom Park ◽

James A Wohlschlegel ◽

...

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthesis ◽

Acyl Carrier Protein ◽

Acyl Chain ◽

Acid Synthesis ◽

Complex Iii ◽

Carrier Protein ◽

Prosthetic Group ◽

Malaria Parasites ◽

Mitochondrial Fatty Acid

Most eukaryotic cells retain a mitochondrial fatty acid synthesis (FASII) pathway whose acyl carrier protein (mACP) and 4-phosphopantetheine (Ppant) prosthetic group provide a soluble scaffold for acyl chain synthesis and biochemically couple FASII activity to mitochondrial electron transport chain (ETC) assembly and Fe-S cluster biogenesis. In contrast, the mitochondrion of Plasmodium falciparum malaria parasites lacks FASII enzymes yet curiously retains a divergent mACP lacking a Ppant group. We report that ligand-dependent knockdown of mACP is lethal to parasites, indicating an essential FASII-independent function. Decyl-ubiquinone rescues parasites temporarily from death, suggesting a dominant dysfunction of the mitochondrial ETC. Biochemical studies reveal that Plasmodium mACP binds and stabilizes the Isd11-Nfs1 complex required for Fe-S cluster biosynthesis, despite lacking the Ppant group required for this association in other eukaryotes, and knockdown of parasite mACP causes loss of Nfs1 and the Rieske Fe-S protein in ETC Complex III. This work reveals that Plasmodium parasites have evolved to decouple mitochondrial Fe-S cluster biogenesis from FASII activity, and this adaptation is a shared metabolic feature of other apicomplexan pathogens, including Toxoplasma and Babesia. This discovery unveils an evolutionary driving force to retain interaction of mitochondrial Fe-S cluster biogenesis with ACP independent of its eponymous function in FASII.

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The mitochondrial acyl carrier protein (ACP) coordinates mitochondrial fatty acid synthesis with iron sulfur cluster biogenesis

eLife ◽

10.7554/elife.17828 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 76

Author(s):

Jonathan G Van Vranken ◽

Mi-Young Jeong ◽

Peng Wei ◽

Yu-Chan Chen ◽

Steven P Gygi ◽

...

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthesis ◽

Acyl Carrier Protein ◽

Acyl Chain ◽

Acid Synthesis ◽

Carrier Protein ◽

Iron Sulfur Cluster ◽

Sulfur Cluster ◽

Iron Sulfur ◽

Mitochondrial Fatty Acid

Mitochondrial fatty acid synthesis (FASII) and iron sulfur cluster (FeS) biogenesis are both vital biosynthetic processes within mitochondria. In this study, we demonstrate that the mitochondrial acyl carrier protein (ACP), which has a well-known role in FASII, plays an unexpected and evolutionarily conserved role in FeS biogenesis. ACP is a stable and essential subunit of the eukaryotic FeS biogenesis complex. In the absence of ACP, the complex is destabilized resulting in a profound depletion of FeS throughout the cell. This role of ACP depends upon its covalently bound 4’-phosphopantetheine (4-PP)-conjugated acyl chain to support maximal cysteine desulfurase activity. Thus, it is likely that ACP is not simply an obligate subunit but also exploits the 4-PP-conjugated acyl chain to coordinate mitochondrial fatty acid and FeS biogenesis.

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Genetic Characterization of Accumulation of Polyhydroxyalkanoate from Styrene in Pseudomonas putida CA-3

Applied and Environmental Microbiology ◽

10.1128/aem.71.8.4380-4387.2005 ◽

2005 ◽

Vol 71 (8) ◽

pp. 4380-4387 ◽

Cited By ~ 30

Author(s):

Niall D. O'Leary ◽

Kevin E. O'Connor ◽

Patrick Ward ◽

Miriam Goff ◽

Alan D. W. Dobson

Keyword(s):

Fatty Acid ◽

Pseudomonas Putida ◽

Fatty Acid Synthesis ◽

De Novo ◽

Acyl Carrier Protein ◽

Functional Characterization ◽

Random Mutagenesis ◽

Acid Synthesis ◽

Sole Source

ABSTRACT Pseudomonas putida CA-3 is capable of accumulating medium-chain-length polyhydroxyalkanoates (MCL-PHAs) when growing on the toxic pollutant styrene as the sole source of carbon and energy. In this study, we report on the molecular characterization of the metabolic pathways involved in this novel bioconversion. With a mini-Tn5 random mutagenesis approach, acetyl-coenzyme A (CoA) was identified as the end product of styrene metabolism in P. putida CA-3. Amplified flanking-region PCR was used to clone functionally expressed phenylacetyl-CoA catabolon genes upstream from the sty operon in P. putida CA-3, previously reported to generate acetyl-CoA moieties from the styrene catabolic intermediate, phenylacetyl-CoA. However, the essential involvement of a (non-phenylacetyl-CoA) catabolon-encoded 3-hydroxyacyl-CoA dehydrogenase is also reported. The link between de novo fatty acid synthesis and PHA monomer accumulation was investigated, and a functionally expressed 3-hydroxyacyl-acyl carrier protein-CoA transacylase (phaG) gene in P. putida CA-3 was identified. The deduced PhaG amino acid sequence shared >99% identity with a transacylase from P. putida KT2440, involved in 3-hydroxyacyl-CoA MCL-PHA monomer sequestration from de novo fatty acid synthesis under inorganic nutrient-limited conditions. Similarly, with P. putida CA-3, maximal phaG expression was observed only under nitrogen limitation, with concomitant PHA accumulation. Thus, β-oxidation and fatty acid de novo synthesis appear to converge in the generation of MCL-PHA monomers from styrene in P. putida CA-3. Cloning and functional characterization of the pha locus, responsible for PHA polymerization/depolymerization is also reported and the significance and future prospects of this novel bioconversion are discussed.

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