Human FAD synthase (isoform 2): a component of the machinery that delivers FAD to apo‐flavoproteins

Abstract

A soluble form of human FAD synthase (isoform 2; hFADS2) was produced and purified to homogeneity as a recombinant His-tagged protein.The enzyme binds 1 mole of the FAD product very tightly, although noncovalently. Complete release of FAD from the 'as isolated' protein requires extensive denaturation. A 75 : 25 mixture of apo ⁄ holoprotein could be prepared by treatment with mild chaotropes, allowing estimatation of the contribution made by bound FAD to the protein stability and evaluatation of whether structural rearrangements may be required for FAD release. Under turnover conditions, the enzyme catalyzes FAD assembly from ATP and FMN and, at a much lower rate, the pyrophosphorolytic hydrolysis of FAD. Several mechanistic features of both reactions were investigated in detail, along with their dependence on environmental conditions (pH, temperature, dependence on metals). Our data indicate that FAD release may represent the rate-limiting step of the whole catalytic cycle and that the process leading to FAD synthesis, and delivery to client apoproteins may be tightly controlled.

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

“…A marked band was immuno-detected in all the fractions containing the over-expressed protein, demonstrating that it corresponded to a domain of hFADS (Figure 3C). The absorbance spectrum of the recombinant Δ 1-328 -hFADS (Figure 3D, straight line) showed a typical flavoprotein absorbance spectrum, similar to that of the entire hFADS2 [15], with a main peak at 275 nm and two minor peaks at ~350 and 450 nm. The FAD/protein monomer ratio estimated in four different protein preparations from the absorption spectrum (Fl%), as described in Section 3.7, was equal to 0.82 ± 0.13, in good agreement with the FAD/protein monomer ratio estimated for the wild-type protein in [15].…”

“…The human cytosolic FADS, hFADS2, was produced and purified to homogeneity as a recombinant His-tagged protein in [15]. 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 hFADS2 monomer.…”

Role of Key Residues at the Flavin Mononucleotide (FMN):Adenylyltransferase Catalytic Site of the Bifunctional Riboflavin Kinase/Flavin Adenine Dinucleotide (FAD) Synthetase from Corynebacterium ammoniagenes

“…where Δ F is the decrease in the value of the fluorescence expressed in arbitrary units, Δ t is the measurement of reaction time expressed in per minute, and K FMN and K FAD are the FMN and FAD fluorescent rate constants expressed in per micromolar [37]. The kinetic data obtained for one substrate at saturated concentrations of the second one were interpreted using the Michaelis-Menten kinetic model.…”

Membrane transporters for the special amino acid glutamine: structure/function relationships and relevance to human health

“…A strategy previously used for OCTN subfamily was adopted for LAT1and 4F2hc over-expression (Indiveri et al, 2013) (Figure 3). After screening of plasmid and cell combinations, the pH6EX3 plasmid revealed suitable for significant over-expression of the protein in E. coli Rosetta DE3pLysS (Brizio et al, 2006; Torchetti et al, 2011). Optimization of some parameters, such as temperature growth and IPTG concentrations (Brizio et al, 2006) was performed and followed by an affinity chromatography procedures (Galluccio et al, 2013).…”