期刊
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
卷 20, 期 24, 页码 -出版社
MDPI
DOI: 10.3390/ijms20246203
关键词
FAD synthase; FMN adenylyl transferase; FADS isoform 6; supermutant
资金
- Fondi di Ateneo the Universita degli Studi di Bari
- Effetto di mutazioni di FLAD1 e di alterazioni dell'omeostasi delle flavine sullo stato redox e sulla biogenesi mitocondriale: uno studio integrato su fibroblasti umani the Universita degli Studi di Bari
- Fondi di Ateneo the Universita della Calabria
- Spanish Agencia Estatal de Investigacion
- Fondo Europeo de Desarrollo Regional, AEI/FEDER, UE [BIO2016-75183-P]
- Government of Aragon-FEDER Grupo de Referencia Biologia Estructural [E35_17R]
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 K-m, while the k(cat) 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.
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