Mutation of aspartate 238 in FAD synthase isoform 6 increases the specific activity by weakening the FAD binding

Leone, Piero ; Galluccio, Michele ; Quarta, Stefano ; Anoz-Carbonell, Ernesto (Universidad de Zaragoza) ; Medina, Milagros (Universidad de Zaragoza) ; Indiveri, Cesare ; Barile, Maria

Resumen: 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.

Idioma: Inglés

DOI: 10.3390/ijms20246203

Año: 2019

Publicado en: International Journal of Molecular Sciences 20, 24 (2019), 6203 [17 pp.]
ISSN: 1661-6596

Factor impacto JCR: 4.556 (2019)

Categ. JCR: CHEMISTRY, MULTIDISCIPLINARY rank: 48 / 177 = 0.271 (2019) - Q2 - T1
Categ. JCR: BIOCHEMISTRY & MOLECULAR BIOLOGY rank: 74 / 297 = 0.249 (2019) - Q1 - T1

Factor impacto SCIMAGO: 1.317 - Medicine (miscellaneous) (Q1) - Physical and Theoretical Chemistry (Q1) - Computer Science Applications (Q1) - Inorganic Chemistry (Q1) - Spectroscopy (Q1) - Organic Chemistry (Q1) - Molecular Biology (Q2) - Catalysis (Q2)

Financiación: info:eu-repo/grantAgreement/ES/DGA/E35-17R
Financiación: info:eu-repo/grantAgreement/ES/MINECO/BIO2016-75183-P

Tipo y forma: Article (Published version)

Área (Departamento): Área Bioquímica y Biolog.Mole. (Dpto. Bioq.Biolog.Mol. Celular)