Figure 3.

DHRSX produces polyprenal from polyprenol using both NAD⁺ and NADP⁺ as cofactor (reaction 1)

(A) Formation of polyprenal from polyprenol was measured after incubation of 5 μg/mL polyprenol with 1 mmol/L NADP⁺ or NAD⁺ and 0.075 μmol/L recombinant DHRSX (see Figure S3A) for 2 h at 37°C. See also Figures S3B, S3C, and S3D.

(B) Kinetic parameters for DHRSX were determined by measuring polyprenal formation after incubation of the indicated concentrations of polyprenol with 1 mmol/L NADP⁺ or NAD⁺ and 0.00375 μmol/L recombinant DHRSX for 5 min at 37°C, or after an identical incubation of 4 μmol/L polyprenol with variable nucleotide concentrations. Data are turnover rates based on formation of polyprenal-18 (means ± SEM; n = 3).

(C) DHRSX is responsible for the polyprenol dehydrogenase activity in HAP1 cells. Polyprenol-18 and polyprenal-18 were monitored after incubation of 1 mg/mL membrane preparations from wild-type (“WT”) and DHRSX KO HAP1 cells with or without polyprenol (5 μg/mL), and NAD⁺ or NADP⁺ (5 mmol/L) for 2 h at 37°C. See also Figure S3E.

(D) Endogenous activity in membrane preparations from EBV-immortalized lymphoblast from controls, parents, and patients was assessed in the reverse direction using NADH or NADPH at 5 mmol/L and polyprenal as substrate. See also Figure S3F.

(E) Correlation of polyprenal reductase activity in EBV-immortalized lymphoblast membrane preparations (Figure 3D) with ß-tubulin-normalized DHRSX protein levels (Figure 1J). Figures 3A, 3C, and 3D present TIC-normalized AUC (means ± SEM; n = 3).

See also Figure S3.