Figure 7.

Resolution by thin layer chromatography of the products of the RFK and the NT activities of selected CaFADS 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.