Bacterial Over-Expression and Purification of the 3'phosphoadenosine 5'phosphosulfate (PAPS) Reductase Domain of Human FAD Synthase: Functional Characterization and Homology Modeling

Abstract

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 (Δ1-328-hFADS) has been over-produced and purified to homogeneity as a recombinant His-tagged protein. The recombinant Δ1-328-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 Δ1-328-hFADS enzyme shows a slight, but not significant, change of Km values (0.24 and 6.23 μM for FMN and ATP, respectively) and of kcat (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 Δ1-328-hFADS versus wild-type protein.

Mutation of Aspartate 238 in FAD Synthase Isoform 6 Increases the Specific Activity by Weakening the FAD Binding

“…Indeed, the extremely low K m for FAD, measured in the course of the reverse reaction, indicates the presence of a very stable bond between FAD. This is in agreement with the finding that size exclusion chromatography is not able to remove FAD from the protein [21,22]. However, the presence of a covalent linkage was previously excluded on the basis of the acidic treatment [23].…”

“…As expected, it is not able to perform FAD hydrolysis [21]. The molecular features of this novel natural form well correlate with those of a previously produced artificial construct, lacking the first 231 residues of hFADS2, which per se can fold and catalyze the FAD synthesis reaction [22]. Therefore, this novel isoform of FAD forming enzymes is, in our opinion, a good model to address remaining challenges in the catalytic behavior of the FAD synthesis reaction in humans, as compared with the our deeper understanding of the yeast orthologues [13,14], from which structure the human protein was modeled [21].…”

Riboflavin transport and metabolism in humans

“…5). Besides the PAPS reductase domain, which is per se able and sufficient to catalyze FAD synthesis starting from FMN and ATP (Miccolis et al 2012), an additional Nterminal located domain is present in the hFADSs characterized up to now. It resembles a molybdo-pterin-binding (MbPt) domain.…”

“…A better knowledge of the structural relationships between the two domains of the protein (Miccolis et al 2012;Giancaspero et al 2015b), as well as of the structural changes associated to the redox switch cysteine-related architecture , might enlighten a number of relevant cellular processes as outlined in Fig. 2.…”

“…The problem of FADS delivery to nascent client apo-enzymes As mentioned above, the purified recombinant hFADS2 behaves as a FAD binding protein, showing a typical flavoprotein absorbance spectrum, with a main peak at 274 nm and two minor peaks at 350 and 450 nm. In its PAPS reductase domain, the enzyme binds one mole of the FAD product very tightly, although non-covalently, with a FAD/protein ratio equal to 0.86 ± 0.2 mol FAD per monomer (Torchetti et al 2011;Miccolis et al 2012). In a number of BclientF ig.…”