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Structure-function analysis of enoyl thioester reductase involved in mitochondrial maintenance
Tomi T Airenne1, Juha M Torkko, Sam Van den plas
1Biocenter Oulu and Department of Biochemistry, P.O. Box 3000, FIN-90014 University of Oulu, Finland.
Journal of Molecular Biology
|March 5, 2003
Summary
The study reveals that mitochondrial fatty acid synthesis (FAS) reductases Etr1p and Mrf1p are crucial for yeast respiratory function. Their activity is essential for maintaining the integrity of the respiratory-competent organelle.
Area of Science:
- Biochemistry
- Molecular Biology
- Yeast Genetics
Background:
- Enoyl thioester reductases (ETRs) are key enzymes in fatty acid synthesis (FAS).
- Prokaryotic ETRs belong to the short-chain dehydrogenases/reductases (SDR) family, while eukaryotic mitochondrial ETRs like Etr1p and Mrf1p belong to the medium-chain dehydrogenases/reductases (MDR) superfamily, suggesting independent evolutionary origins.
- Mitochondrial FAS plays a vital role in cellular respiration.
Purpose of the Study:
- To investigate the structural and functional roles of Candida tropicalis enoyl thioester reductase Etr1p and its Saccharomyces cerevisiae homologue Mrf1p.
- To elucidate the significance of mitochondrial enoyl thioester reductase activity for yeast respiratory function.
- To determine the evolutionary relationship between prokaryotic and eukaryotic ETRs.
Main Methods:
- Crystallography was used to determine the structures of Etr1p, its complex with NADPH, and a key mutant (Etr1Y79Np).
- Site-directed mutagenesis was employed to create Etr1Y79Np and a corresponding mutant in Mrf1p (Tyr73).
- Functional assays were performed to assess enzyme activity and rescue of respiratory deficiency in yeast strains.
Main Results:
- The crystal structures revealed that Etr1p and Mrf1p are structurally distinct from prokaryotic ETRs and belong to the MDR superfamily.
- The Etr1Y79Np mutant retained its native fold but exhibited significantly reduced catalytic activity (0.1% of wild-type).
- Mutagenesis of the corresponding Tyr73 residue in Mrf1p yielded similar loss-of-function results, and the mutant failed to rescue respiratory deficiency in mrf1Δ yeast.
Conclusions:
- Mitochondrial enoyl thioester reductase activity, mediated by Mrf1p/Etr1p, is indispensable for yeast respiratory function.
- The study highlights the independent evolution of two distinct classes of ETRs.
- Mitochondrial FAS is critical for the integrity and function of the respiratory-competent organelle in yeast.