Figure S5.

Additional data supporting that DHRSX also converts dolichal to dolichol, related to Figure 5

(A) Representative extracted ion chromatograms of the forward reaction (dolichal to dolichol conversion; left side) and reverse reaction (dolichol to dolichal conversion; right side) of DHRSX presented in Figures 5A and S5B. Metabolites were assessed after incubation of 5 μg/mL dolichal or dolichol with 1 mmol/L of the indicated nucleotides and 0.075 μmol/L recombinant DHRSX for 2 h at 37°C.

(B) Formation of dolichal from dolichol was assessed after incubation of 5 μg/mL dolichol with 0.075 μmol/L recombinant DHRSX, and 1 mmol/L NAD(P)⁺, 2h, 37°C. Data is TIC-normalized AUC of 18 isoprenoid unit containing lipids (means ± SEM, n = 3). See Figure 5A for forward reaction.

(C and D) Lack of specificity of DHRSX for NAD(H) or NADP(H) in the conversion of polyprenol to polyprenal, as well as in the conversion of dolichal to dolichol in forward and reverse direction. Polyprenol-18 (POL), polyprenal-18 (PAL), dolichol-18 (DOL) or dolichal-18 (DAL) were measured at the indicated timepoints after incubation of 0.075 μmol/L recombinant DHRSX protein with 5 μg/mL of POL, PAL, DOL or DAL and the indicated cofactor concentrations at 37°C. Data are represented as mean TIC-normalized AUC of three replicates ±SEM. Panels showing bidirectional polyprenol to polyprenal conversion have already been shown in Figure S3D and are displayed here to facilitate a comparison. Of note, the progression of these reactions with time indicates that conversion of dolichol to dolichal is much less favorable than the conversion of polyprenol to polyprenal, consistent with prior reports that the presence of a double-bond between C2 and C3 (as present in polyprenol) strongly favors the oxidation of a terminal alcohol group by increasing the equilibrium constant by a factor of more than 100).³⁰.