Characterization of mitochondrial 2-enoyl thioester reductase involved in respiratory competence
1University of Oulu, Biocenter Oulu
2University of Oulu, Faculty of Science, Department of Biochemistry
|Online Access:||PDF Full Text (PDF, 1 MB)|
|Persistent link:|| http://urn.fi/urn:isbn:9514270312
|Publish Date:|| 2003-05-23
|Thesis type:||Doctoral Dissertation
|Defence Note:||Academic Dissertation to be presented with the assent of the Faculty of Science, University of Oulu, for public discussion in Raahensali (Auditorium L10), Linnanmaa, on May 23rd, 2003, at 12 noon.
Docent Sirkka Keränen
Docent Pentti Somerharju
Maintenance of the mitochondrial respiratory chain complexes plays crucial role for the aerobic metabolism of the eukaryotes such as unicellular yeasts, for example, Saccharomyces cerevisiae as well as of human being.
Mitochondrial respiratory function has been studied using the yeast S. cerevisiae as a model organism. Since yeast cells are also able to grow without respiration by fermentation, identification of the nuclear genes linked to respiratory function is possible by generation of nuclear gene deletions and testing for respiration-deficient phenotype of the yeast deletion strains id est for yeast cells only poorly or not at all growing on the media containing non-fermentable carbon sources.
This study reports identification of a novel mitochondrial 2-enoyl thioester reductase from the yeasts Candida tropicalis and S. cerevisiae, Etr1p and Mrf1p, respectively. Examination of the function of these proteins in the respiration-deficient mrf1Δ strain from S. cerevisiae suggests that the reductase is involved in mitochondrial fatty acid synthesis (FAS type II) in the yeast. Site-directed mutagenesis of a conserved tyrosine in the catalytic site of the enzyme indicated that the 2-enoyl thioester reductase activity is critical for mitochondrial respiratory competence. In addition, subcellular localization to mitochondria was required for the complementation of the respiration-deficient phenotype of the yeast reductase deletion strain. The crystal structure for the Etr catalytic site mutant indicated the structural integrity of the mutant supporting the requirement of the tyrosine for the catalysis.
Characterization of Etr crystal structures both in apo- and holo-forms containing NADPH established Etr as a member of novel subfamily of enoyl thioester reductases in the superfamily of medium-chain dehydrogenases/reductases (MDR). Two isoforms of Etr with the difference in three amino acids only are encoded by two distinct genes in C. tropicalis, whereas only single gene encodes the reductase functioning in the mitochondria in S. cerevisiae. The presence of two genes in C. tropicalis was taken as an example of genetic redundancy in this yeast, the two genes also shown to be expressed in slightly different ways under various carbon sources available for growth.
Acta Universitatis Ouluensis. A, Scientiae rerum naturalium
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