Abstract
FAD synthase (FADS, EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor, FAD. Human FADS is organized in two domains: -the 3′phosphoadenosine 5′phosphosulfate (PAPS) reductase domain, similar to yeast Fad1p, at the C-terminus, and -the resembling molybdopterin-binding domain at the N-terminus. To understand whether the PAPS reductase domain of hFADS is sufficient to catalyze FAD synthesis, per se, and to investigate the role of the molybdopterin-binding domain, a soluble “truncated” form of hFADS lacking the N-terminal domain (Δ1-328-hFADS) has been over-produced and purified to homogeneity as a recombinant His-tagged protein. The recombinant Δ1-328-hFADS binds one mole of FAD product very tightly as the wild-type enzyme. Under turnover conditions, it catalyzes FAD assembly from ATP and FMN and, at a much lower rate, FAD pyrophosphorolytic hydrolysis. The Δ1-328-hFADS enzyme shows a slight, but not significant, change of Km values (0.24 and 6.23 μM for FMN and ATP, respectively) and of kcat (4.2 × 10−2 s−1) compared to wild-type protein in the forward direction. These results demonstrate that the molybdopterin-binding domain is not strictly required for catalysis. Its regulatory role is discussed in light of changes in divalent cations sensitivity of the Δ1-328-hFADS versus wild-type protein.
Highlights
The primary role of the water-soluble vitamin B2, i.e., riboflavin (Rf), in cell biology is connected with its conversion into FMN and FAD, the cofactors of a large number of dehydrogenases, reductases and oxidases involved in energetic metabolism, redox homeostasis and protein folding, as well as in diverse regulatory events [1,2,3]
FMN, a multi-alignment was performed among the hFADS1, FMN adenylyltransferase from C. glabrata and Fad1p from S. cerevisiae
The folding of the phosphoadenosine 5'phosphosulfate (PAPS) reductase domain resembles those of the related enzymes FMNAT of C. glabrata and Fad1p from S. cerevisiae (Figure 2B,C), in agreement with the rmsd value of 1.10 Å for 163 superimposed Cα atoms of the PAPS reductase domain on FMNAT of C. glabrata, calculated by Swiss-PdbViewer 4.0.1
Summary
The primary role of the water-soluble vitamin B2, i.e., riboflavin (Rf), in cell biology is connected with its conversion into FMN and FAD, the cofactors of a large number of dehydrogenases, reductases and oxidases involved in energetic metabolism, redox homeostasis and protein folding, as well as in diverse regulatory events [1,2,3]. Two enzymes are required for flavin cofactor synthesis starting from the vitamin: Rf kinase (RFK, ATP:riboflavin 5' phosphotransferase, EC 2.7.1.26), which transfers a phosphoryl group from ATP to Rf to form FMN, and FMN adenylyl transferase (FMNAT, ATP: FMN adenylyl transferase, EC 2.7.7.2), that adenylates FMN to give FAD. Fmn1p shows sequence and structure similarity to the RFK-module of prokaryotic FADS and appears largely conserved through evolution. FADS is interesting as a potential target for the development of novel antimicrobial drugs [10,11]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
