Succinate dehydrogenase flavoprotein subunit expression in Saccharomyces cerevisiae--involvement of the mitochondrial FAD transporter, Flx1p.

The mitochondrial FAD transporter, Flx1p, is a member of the mitochondrial carrier family responsible for FAD transport in Saccharomyces cerevisiae. It has also been suggested that it has a role in maintaining the normal activity of mitochondrial FAD-binding enzymes, including lipoamide dehydrogenase and succinate dehydrogenase flavoprotein subunit Sdh1p. A decrease in the amount of Sdh1p in the flx1Delta mutant strain has been determined here to be due to a post-transcriptional control that involves regulatory sequences located upstream of the SDH1 coding sequence. The SDH1 coding sequence and the regulatory sequences located downstream of the SDH1 coding region, as well as protein import and cofactor attachment, seem to be not involved in the decrease in the amount of protein

Expression of succinate dehydrogenase flavoprotein subunit in saccharomyces cerevisiae studied by lacZ reporter strategy. Effect of FLX1 deletion.

We described here the construction of two novel Saccharomyces cerevisiae strains in which the regulatory region of the SDH1 gene, coding for the succinate dehydrogenase flavoprotein subunit, was fused in frame to the reporter gene lacZ of E. coli, coding for beta-galactosidase. By this approach, SDH1 expression was studied in the yeast strain, flx1 delta-lacZ, lacking of a functional mitochondrial FAD translocator, Flx1p. The experiments described here are in line with the hypotesys that a correlation exists between defects in flavin cofactor homeostasis and mitochondrial apo-flavoprotein expression

Bacterial over-expression and purification of the PAPS reductase domain of human FAD synthase. Functional characterization and homology modelling.

FAD synthase (FADS, EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor, FAD. Human FADS is organized in two domains: -the 3′phosphoadenosine 5′phosphosulfate (PAPS) reductase domain, similar to yeast Fad1p, at the C-terminus, and -the resembling molybdopterin-binding domain at the N-terminus. To understand whether the PAPS reductase domain of hFADS is sufficient to catalyze FAD synthesis, per se, and to investigate the role of the molybdopterin-binding domain, a soluble “truncated” form of hFADS lacking the N-terminal domain (Δ(1-328)-hFADS) has been over-produced and purified to homogeneity as a recombinant His-tagged protein. The recombinant Δ(1-328)-hFADS binds one mole of FAD product very tightly as the wild-type enzyme. Under turnover conditions, it catalyzes FAD assembly from ATP and FMN and, at a much lower rate, FAD pyrophosphorolytic hydrolysis. The Δ(1-328)-hFADS enzyme shows a slight, but not significant, change of K(m) values (0.24 and 6.23 μM for FMN and ATP, respectively) and of k(cat) (4.2 × 10(−2) s(−1)) compared to wild-type protein in the forward direction. These results demonstrate that the molybdopterin-binding domain is not strictly required for catalysis. Its regulatory role is discussed in light of changes in divalent cations sensitivity of the Δ(1-328)-hFADS versus wild-type protein

Mitochondrial localization of human FAD synthetase isoform 1

FAD synthetase or ATP:FMN adenylyl transferase (FADS or FMNAT, EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. We face here the still controversial sub-cellular localization of FADS in eukaryotes. First, by western blotting experiments, we confirm the existence in rat liver of different FADS isoforms which are distinct for molecular mass and sub-cellular localization. A cross-reactive band with an apparent molecular mass of 60 kDa on SDS–PAGE is localized in the internal compartments of freshly isolated purified rat liver mitochondria. Recently we have identified two isoforms of FADS in humans, that differ for an extra-sequence of 97 amino acids at the N-terminus, present only in isoform 1 (hFADS1). The first 17 residues of hFADS1 represent a cleavable mitochondrial targeting sequence (by Target-P prediction). The recombinant hFADS1 produced in Escherichia coli showed apparent Km and Vmax values for FMN equal to 1.3 ± 0.7 lM and 4.4 ± 1.3 nmol min1 mg protein1, respectively, and was inhibited by FMN at concentration higher than 1.5 lM. The in vitro synthesized hFADS1, but not hFADS2, is imported into rat liver mitochondria and processed into a lower molecular mass protein product. Immunofluorescence confocal microscopy performed on BHK-21 and Caco-2 cell lines transiently expressing the two human isoforms, definitively confirmed that hFADS1, but not hFADS2, localizes in mitochondria