Fig. 5: Biochemical and kinetic characterization of the recombinant G3PDH isoenzymes Saci_1118 and Saci_2032.

a Absorption spectrum (400–500 nm) of Saci_2032 (blue solid line) and Saci_1118 (red solid line) at 70°C with bound FAD before addition of G3P. After addition of G3P (10 min), the loss of absorption at 450 nm indicates the G3P-dependent reduction of bound FAD in Saci_1118 (red dashed line) and Saci_2032 (blue dashed line). The maximal absorption at 450 nm after denaturation by SDS was used to calculate the amount of bound FAD per protein. b The kinetic properties of recombinant Saci_1118 (red circles) and Saci_2032 (blue squares) with G3P as substrate (0–0.3 mM) were determined in a continuous assay at 70 °C by coupling the oxidation of G3P to the reduction of the artificial redox active dye DCPIP following the decrease of absorbance at 600 nm. c Absorption spectra of ubiquinone-Q1 at 70 °C in the absence (solid line) and in the presence of Saci_1118 (red dotted line) or Saci_2032 (blue dashed line) and 200 µM of G3P after 60 seconds. Loss of absorption at 280 nm indicates reduction of ubiquinone-Q1 by G3PDH. d The kinetic properties of Saci_1118 (red circle) and Saci_2032 (blue square) with respect to ubiquinone-Q1 were determined at 70 °C in a continuous assay following the G3P-dependent reduction of ubiquinone-Q1 to ubiquinol-Q1 at 280 nm. Experiments were performed in triplicate (n = 3, technical replicates) and error bars indicate the SD of the mean and individual data points are shown as grey dots.