Figure 7From: Structural analysis of FAD synthetase from Corynebacterium ammoniagenesResolution by thin layer chromatography of the products of the RFK and the NT activities of selected Ca FADS variants. The reaction mixtures contained: (A), 2-WT and 50 μM RF, 3-H28D and 50 μM RF, 4-R161D and 50 μM RF, 5-S164D and 50 μM RF, 6-T208A and 50 μM RF, 7-T208A and 50 μM FMN and 8-E268D and 50 μM RF. (B), 2-WT CaFADS and 50 μM RF, 3-CaFADS C-terminal domain and 50 μM RF, 4-CaFADS C-terminal domain and 50 μM FMN and 5-a reaction mixture containing RF incubated in the absence of enzyme. Lanes (1) in (A) and (B) corresponded to a standard solution containing 50 μM RF, 50 μM FMN and 50 μM FAD. All the reaction mixtures contained 0.2 mM ATP and 10 mM MgCl2 in 50 mM Tris/HCl, pH 8.0. Experiments in (A) were carried out with pure CaFADS variants at concentration 1 μM, while reaction mixtures in (B) contained crude extracts with the over-expressed CaFADS C-terminal domain. (C) Product formation in the assayed samples. X, T and hyphen indicate production, detection of only traces and no production of the corresponding reaction product, respectively.Back to article page