INVESTIGADORES
NUSBLAT alejandro David
congresos y reuniones científicas
Título:
Cyt b5 dependent-C5(6) sterol desaturase from Tetrahymena thermophila complements ergosterol biosynthesis in Sacharomyces cerevisiae
Autor/es:
MAURO RINALDI; MARIELA L. TOMAZIC; TOMAS POKLEPOVICH; ALEJANDRO NUSBLAT; CLARA B. NUDEL
Lugar:
San Miguel de Tucumán, Tucumán, Argentina
Reunión:
Congreso; VII CONGRESO ARGENTINO DE MICROBIOLOGÍA GENERAL SAMIGE DEL BICENTENARIO (SAMIGE); 2011
Institución organizadora:
Sociedad Argentina de Microbiología General
Resumen:
Biosynthesis of sterols involves several
enzymatic steps, such as squalene cyclization, demethylations, reductions and
desaturations.
C-5(6) sterol desaturases are important
enzymes in sterol-synthesizing organisms and catalyze the introduction of D5 double bound.
Previous
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
fatty acid hydroxylase superfamily (FAHS).
Although the ciliate Tetrahymena thermophila can not synthesize sterols, it 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
group.
We have recently identified two of them, C-5(6)
sterol desaturase, DES5A,
and C-24 sterol desaturase-like, DES24. Moreover, previous
characterization of sterol desaturases of T.thermophila has
revealed the typical features of these enzymes. The C5(6) sterol desaturase of
yeasts is an endoplasmic reticulum enzyme encoded by ERG3 gene, its disruption creates the interruption of ergosterol synthesis.
In order
to evaluate the functionality of cytochrome b5 dependent enzymes of the ciliate
and to confirm the activity of C-5(6) sterol desaturase, Des5Ap, we performed a heterologous expression in ERG3 deficient strain, erg3-.
Thus, erg3- 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 for the first time that Cyt b5 dependent enzyme of the FAHS of the
ciliate is active in S. cerevisiae, indicating that yeasts
has the necessary requirements to restore the activity with a foreign gene.
Moreover, these data suggest that S.
cerevisiae is a good system to be
used for both the study of membrane protein complexes of FAHS and the identification of putative sterol related
genes of ciliates, such as the C-7(8) sterol desaturase which has
biotechnological applications.