NUSBLAT alejandro David
congresos y reuniones científicas
Heterologous expression of C5(6) sterol desaturase from Tetrahymena thermophila restores ergosterol biosynthesis in Sacharomyces cerevisiae ERG3 knockout strain
TOMAS POKLEPOVICH; MARIELA TOMAZIC; MAURO RINALDI; ALEJANDRO NUSBLAT; CLARA NUDEL
Congreso; European Congress of Protistology; 2011
German Society of Protozoology / Federation of European Protistological Societies -
Sterol biosynthesis comprises various enzymatic steps, such as squalene cyclization, demethylations, reductions and desaturations. Desaturases are essential enzymes in sterol biosynthesis, such as C-5(6) sterol desaturase that catalyze the introduction of Δ5 double bound. Several studies in plants, mammals and yeasts have shown that desaturation at C-5(6) involves an electron transfer from NADH to the terminal oxidase (the desaturase itself) via a cytochrome b5 reductase and the cytochrome b5. These features are typical of the fatty acid hydroxylase superfamily (FAHS). Even though the ciliate Tetrahymena thermophila can not synthesize sterols, performs several modifications in the sterol moiety. Four activities have been described: C-5(6), C-7(8) and C-22(23) sterol desaturations and removal of C-24 ethyl. We have recently identified and characterized two sterol desaturases of T. thermophila, which revealed the typical features of these enzymes: C-5(6) sterol desaturase, DES5A, and C-24 sterol desaturase-like, DES24. In yeast, the C5(6) sterol desaturase is an endoplasmic reticulum enzyme encoded by the ERG3 gene, its disruption creates an interruption in the synthesis of ergosterol. A heterologous expression of Des5Ap in the ERG3 deficient strain was performed in order to evaluate the functionality of cytochrome b5 dependent enzyme of the ciliate and to confirm its activity. The erg3 mutant was transformed with a p425-C5T plasmid, carrying DES5A gene of Tetrahymena. The sterol profile of the complemented strain was analyzed by HPLC and GC-MS. The results showed that complementation restored ergosterol synthesis. Therefore, this work shows by the first time that a ciliate Cyt b5 dependent enzyme of the FAHS is active in Saccharomyces cerevisiae, indicating that yeast has the requirements to restore the activity with a foreign gene. These data suggest that S. cerevisiae can be used as a good system for both the study of membrane protein complex of the FAHS, and the identification of putative genes of ciliates.