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

Maria Barile,Ernesto Anoz-Carbonell,Stefano Quarta,Cesare Indiveri,Milagros Medina,Piero Leone,Michele Galluccio

doi:10.3390/ijms20246203

Abstract

FAD synthase (FADS, or FMN:ATP adenylyl transferase) coded by the FLAD1 gene is the last enzyme in the pathway of FAD synthesis. The mitochondrial isoform 1 and the cytosolic isoform 2 are characterized by the following two domains: the C-terminal PAPS domain (FADSy) performing FAD synthesis and pyrophosphorolysis; the N-terminal molybdopterin-binding domain (FADHy) performing a Co++/K+-dependent FAD hydrolysis. Mutations in FLAD1 gene are responsible for riboflavin responsive and non-responsive multiple acyl-CoA dehydrogenases and combined respiratory chain deficiency. In patients harboring frameshift mutations, a shorter isoform (hFADS6) containing the sole FADSy domain is produced representing an emergency protein. With the aim to ameliorate its function we planned to obtain an engineered more efficient hFADS6. Thus, the D238A mutant, resembling the D181A FMNAT “supermutant” of C. glabrata, was overproduced and purified. Kinetic analysis of this enzyme highlighted a general increase of Km, while the kcat was two-fold higher than that of WT. The data suggest that the FAD synthesis rate can be increased. Additional modifications could be performed to further improve the synthesis of FAD. These results correlate with previous data produced in our laboratory, and point towards the following proposals (i) FAD release is the rate limiting step of the catalytic cycle and (ii) ATP and FMN binding sites are synergistically connected.

Highlights

The riboflavin (Rf) derived FMN and FAD cofactors play a pivotal role in cell economy ensuring the functionality of the flavoproteome, mainly localized in mitochondria [1,2]
The only gene identified for coding functional FAD synthases in humans is FLAD1 gene
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Summary

Introduction

The riboflavin (Rf) derived FMN and FAD cofactors play a pivotal role in cell economy ensuring the functionality of the flavoproteome, mainly localized in mitochondria [1,2]. Consistent with the crucial role of flavins and flavoenzymes in cell life, several diseases, including neuromuscular and neurological disorders are linked to flavin-dependent enzyme deficiency or impairment in Rf homeostasis in humans and experimental animals. These disorders, in some cases, can be cured with high doses of Rf, as the two Rf-responsive (RR) disorders Brown–Vialetto van Laere syndrome (BVVLS) [3,4] and RR-multiple acyl-CoA dehydrogenase deficiency (RR-MADD) [5,6,7]. The only gene identified for coding functional FAD synthases in humans is FLAD1 gene The structures of FADSs from yeast, but not that of the human orthologue, have been solved [13,14]

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Conclusion