FAD synthase (FADS or FMN:ATP adenylyl transferase) is the last enzyme in the pathway converting Riboflavin into the redox cofactor FAD, essential for the activity of hundreds of flavoenzymes. In humans FADS exists in different isoforms generated by different transcript variants of the FLAD1 gene. The longer transcripts code for the mitochondrial isoform 1 and the cytosolic isoform 2. Both the isoforms are bi-functional enzymes containing two domains: a PAPS domain at the C-terminus able to catalyze FAD synthesis (EC 2.7.7.2) and the reverse reaction, i.e. pyrophosphorolysis; a molybdopterin-binding domain at the N-terminus performing a Co++/K+-dependent FAD hydrolysis (EC 3.6.1.18)1. Mutations in FLAD1 gene are responsible for Riboflavin-Responsive and Non-Responsive Multiple Acyl-CoA Dehydrogenases and Combined Respiratory-Chain Deficiency. The survival of patients suffering for frameshift mutations led to the discovery of a novel FLAD1 transcript2, coding for a shorter isoform 6 consisting of the sole PAPS domain, able to synthesize FAD, in a Mg++ dependent manner. Therefore, the shorter isoform could represent a target for therapy intervention in patients harboring FADS defects3. In the aim to choose a strategy ameliorating FAD production in patients, a variant of hFADS6 carrying the site-directed mutation D238A was over-produced and purified by affinity chromatography. The rationale for this approach is that this mutant is expected to exhibit a higher rate of FAD synthesis, as already observed in the orthologue enzyme of Candida glabrata, i.e. FMNAT4. Kinetic analysis allowed to describe some functional features of this enzyme and to prove that the Kcat of the 6His-hFADS6-D238A is (0.10 s-1) about 2-fold higher than that of wt (0.05 s-1). Nevertheless, the KmFMN (1.3 µM) and the KmATP (44.3 µM) of the mutant, as well as the KmFAD (0.04 µM) in the reverse reaction also significantly increase, thus resulting in a decrease of the catalytic efficiency of this enzyme in vitro. This data suggests that additional or alternative strategies are needed to actually increase the emergency action of hFADS6. These results, together with other previously produced in our laboratory, point towards the proposal that i) FAD release is the limiting step of the catalytic cycle ii) ATP and FMN binding sites are sinergically connected.

hFADS6 and its “supermutant”: a possible therapeutic target?

Piero Leone
;
C. Indiveri;M. Barile
2019

Abstract

FAD synthase (FADS or FMN:ATP adenylyl transferase) is the last enzyme in the pathway converting Riboflavin into the redox cofactor FAD, essential for the activity of hundreds of flavoenzymes. In humans FADS exists in different isoforms generated by different transcript variants of the FLAD1 gene. The longer transcripts code for the mitochondrial isoform 1 and the cytosolic isoform 2. Both the isoforms are bi-functional enzymes containing two domains: a PAPS domain at the C-terminus able to catalyze FAD synthesis (EC 2.7.7.2) and the reverse reaction, i.e. pyrophosphorolysis; a molybdopterin-binding domain at the N-terminus performing a Co++/K+-dependent FAD hydrolysis (EC 3.6.1.18)1. Mutations in FLAD1 gene are responsible for Riboflavin-Responsive and Non-Responsive Multiple Acyl-CoA Dehydrogenases and Combined Respiratory-Chain Deficiency. The survival of patients suffering for frameshift mutations led to the discovery of a novel FLAD1 transcript2, coding for a shorter isoform 6 consisting of the sole PAPS domain, able to synthesize FAD, in a Mg++ dependent manner. Therefore, the shorter isoform could represent a target for therapy intervention in patients harboring FADS defects3. In the aim to choose a strategy ameliorating FAD production in patients, a variant of hFADS6 carrying the site-directed mutation D238A was over-produced and purified by affinity chromatography. The rationale for this approach is that this mutant is expected to exhibit a higher rate of FAD synthesis, as already observed in the orthologue enzyme of Candida glabrata, i.e. FMNAT4. Kinetic analysis allowed to describe some functional features of this enzyme and to prove that the Kcat of the 6His-hFADS6-D238A is (0.10 s-1) about 2-fold higher than that of wt (0.05 s-1). Nevertheless, the KmFMN (1.3 µM) and the KmATP (44.3 µM) of the mutant, as well as the KmFAD (0.04 µM) in the reverse reaction also significantly increase, thus resulting in a decrease of the catalytic efficiency of this enzyme in vitro. This data suggests that additional or alternative strategies are needed to actually increase the emergency action of hFADS6. These results, together with other previously produced in our laboratory, point towards the proposal that i) FAD release is the limiting step of the catalytic cycle ii) ATP and FMN binding sites are sinergically connected.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11586/347823
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