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

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.

Insights into the FMNAT Active Site of FAD Synthase: Aromaticity Is Essential for Flavin Binding and Catalysis

“…Thus, the environment around the isoalloxzine is more hydrophobic in the prokaryotic enzyme, while highly conserved charged residues contribute to the isoalloxazine site in the eukaryotic counterparts (Figures 1c and 2). Some of these charged residues, such as D181 in CgFADS and the equivalent D408 in HsFADS, are crucial residues that contribute to the strong binding of the isoalloxazine of flavins, both as substrates and products [6,18,35]. In this way, the eukaryotic enzymes modulate their specific activity and the flavin homeostasis, making the release of the FAD product to the client-apoprotein the limiting step of the reaction [18,36].…”

“…Some of these charged residues, such as D181 in CgFADS and the equivalent D408 in HsFADS, are crucial residues that contribute to the strong binding of the isoalloxazine of flavins, both as substrates and products [6,18,35]. In this way, the eukaryotic enzymes modulate their specific activity and the flavin homeostasis, making the release of the FAD product to the client-apoprotein the limiting step of the reaction [18,36].…”

“…Two isoforms lack the PAPS reductase module while the last isoform discovered lacks the FAD hydrolase module. In addition, these isoforms also differ in subcellular location as well as in catalytic efficiencies and ions requirements [13][14][15][16][17][18]. There are no crystallographic structures available for any of these HsFADSs, but some structural models have been built [17,19,20].…”

Development of Novel Experimental Models to Study Flavoproteome Alterations in Human Neuromuscular Diseases: The Effect of Rf Therapy

“…Both hFADS1 and 2 contain an N-terminal molybdopterin binding (MPTb, recently renamed FADHy [ 35 ] domain, since it hides a FAD hydrolase activity [ 34 , 35 ], which is fused with a C-terminal 3-phosphoadenosine 5-phosphosulfate reductase domain (PAPS, recently renamed FADSy [ 35 ]), which per se performs the FADS activity [ 73 , 74 , 75 ].…”

“…The two functional domains of the longest isoforms are colored in the background. Homology modeling of the single domains are reported in [ 7 , 34 , 73 , 74 ].…”