In Saccharomyces cerevisiae, the cytosolic and promitochondrial isoenzymes of fumarate reductase are encoded by the FRDS and OSM1 genes, respectively. The product of the OSM1 gene is reported to be required for growth in hypertonic medium. Simultaneous disruption of the FRDS and OSM1 genes resulted in the inability of the yeasts to grow anaerobically on glucose as a carbon source, and disruption of the OSM1 gene caused poor growth under anaerobic conditions. However, the disruption of both the FRDS and/or OSM1 genes had no effect on aerobic growth or growth under hypertonic conditions. These results suggest that the fumarate reductase isoenzymes in Saccharomyces cerevisiae are essential for anaerobic growth but not for growth under hypertonic conditions.
A gene of the soluble fumarate reductase (FRDS) that binds FAD non-covalently was cloned by polymerase chain reaction (PCR) using degenerate oligonucleotides designed from partial amino acid sequences of highly purified enzyme. The nucleotide sequence of a 0.99-kb amplified product was found to be nearly identical to a partial sequence of an open reading frame (ORF) previously reported (EMBL database accession number S-30830). According to the sequence in the EMBL database, we cloned 1.7-kb fragment containing entire sequence of this ORF by PCR and found that this fragment contained a perfect match to the 0.99-kb sequence amplified with the degenerate primers. From these results, we concluded that this ORF is the FRDS gene. The amino acid sequences of the regions involved in the non-covalent binding of FAD and the active site, which are conserved among the flavoprotein subunits of membrane-bound fumarate reductase and succinate dehydrogenase, were found in FRDS. However, unlike the membrane-bound enzymes, FRDS did not contain the histidine residue that covalently binds the isoalloxazine ring of FAD at or near the corresponding position. FRDS showed high homology to the product of S. cerevisiae OSM1 gene which was reported to be required for growth in hypertonic media.
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