87593 20201130083156.0 doi 10.3390/ijms20246203 sideral 115804 ART-2019-115804 eng Leone, Piero Mutation of aspartate 238 in FAD synthase isoform 6 increases the specific activity by weakening the FAD binding 2019 Access copy available to the general public Unrestricted 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. info:eu-repo/grantAgreement/ES/DGA/E35-17R info:eu-repo/grantAgreement/ES/MINECO/BIO2016-75183-P info:eu-repo/semantics/openAccess by http://creativecommons.org/licenses/by/3.0/es/ 4.556 2019 CHEMISTRY, MULTIDISCIPLINARY 48 / 177 = 0.271 2019 Q2 T1 BIOCHEMISTRY & MOLECULAR BIOLOGY 74 / 297 = 0.249 2019 Q1 T1 1.317 2019 Medicine (miscellaneous) 2019 Q1 Physical and Theoretical Chemistry 2019 Q1 Computer Science Applications 2019 Q1 Inorganic Chemistry 2019 Q1 Spectroscopy 2019 Q1 Organic Chemistry 2019 Q1 Molecular Biology 2019 Q2 Catalysis 2019 Q2 info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion Galluccio, Michele Quarta, Stefano (orcid)0000-0002-6649-9153 Anoz-Carbonell, Ernesto Universidad de Zaragoza (orcid)0000-0001-8743-0182 Medina, Milagros Universidad de Zaragoza Indiveri, Cesare Barile, Maria 1002 060 Universidad de Zaragoza Dpto. Bioq.Biolog.Mol. Celular Área Bioquímica y Biolog.Mole. 20, 24 (2019), 6203 [17 pp.] Int. j. mol. sci. International Journal of Molecular Sciences 1661-6596 3269024 http://zaguan.unizar.es/record/87593/files/texto_completo.pdf Versión publicada 108494 http://zaguan.unizar.es/record/87593/files/texto_completo.jpg?subformat=icon icon Versión publicada oai:zaguan.unizar.es:87593 articulos driver 2020-11-30-07:58:07 ARTICLE