Figure S3.

Additional data supporting that DHRSX converts polyprenol to polyprenal, related to Figure 3

(A) Immunoblot using an antibody against the 6xHis epitope of recombinant DHRSX-N-His, alongside Ponceau stain of the same membrane.

(B) Adjunct to Figures 3A and S3C, representative chromatograms of the forward reaction (polyprenol to polyprenal conversion; left side) and reverse reaction (polyprenal to polyprenol conversion; right side) with recombinant DHRSX. Interconversion was measured after incubation of 5 μg/mL of polyprenol or polyprenal with 1 mmol/L of the indicated cofactor, and 0.075 μmol/L recombinant DHRSX for 2 h at 37°C.

(C) Polyprenol formation from polyprenal was measured after incubation of 5 μg/mL polyprenal with 1 mmol/L NADPH or NADH and 0.075 μmol/L recombinant DHRSX for 2 h at 37°C. Measurements are based on the formation of polyprenol with 18 isoprene units.

(D) Time course of polyprenal/polyprenol interconversion by recombinant DHRSX and dependence on NAD(P)(H) concentration measured in both directions. Polyprenol-18 and polyprenal-18 were measured at the indicated time-points after incubation of 5 μg/mL of polyprenol with the indicated concentrations of NADP⁺ or NAD⁺, or of 5 μg/mL of polyprenal with the indicated concentrations of NADPH or NADH and 0.075 μmol/L recombinant DHRSX (see Figure S3A) at 37°C.

(E) Adjunct to Figure 3C showing the reverse activity of DHRSX measured in WT and DHRSX KO HAP1 cell membrane extracts. Polyprenol-18 and polyprenal-18 were quantified after incubation of 1 mg/mL HAP1 membrane extracts with/without polyprenal (5 μg/mL) and NADH or NADPH (5 mmol/L), 2h, 37°C.

(F) Measurement of dolichal-18 from the experiment presented in Figure 3D. Isoprenoid species were monitored after incubation of 1 mg/mL EBV-immortalized lymphoblast membrane extracts with/without polyprenal (5 μg/mL) and NADH or NADPH (5 mmol/L), 2h, 37°C. Figures S3C, S3D, S3E and S3F present TIC-normalized AUC (means +/− SEM; n = 3).