Catalytic modulation of human cytochromes P450 17A1 and P450 11B2 by phospholipid

Abstract

Unlike most of the drug-metabolizing cytochrome P450s, microsomal P450 17A1 and mitochondrial P450 11B2 catalyze sequential multi-step reactions in steroid biosynthesis. The membrane phospholipid composition might be one parameter that modulates the efficiency and processivity of specific pathways. Here we systematically examined the effects of physiologically relevant phospholipids on the catalysis of purified P450 17A1, P450 11B2, and P450 11B1 in reconstituted assay systems. Both dioleoylphosphatidylcholine (DOPC, 18:1) and dilauroylphosphatidylcholine (DLPC, 12:0) were found to be very efficient in reconstituting 17-hydroxylase and 1720-lyase reactions of P450 17A1. Phosphatidylethanolamine (PE) specifically enhanced 1720-lyase activity up to 2.4-fold in the presence of phosphatidylcholine. On the other hand, P450 11B2-catalyzed production of aldosterone from 11-deoxycorticosterone was very low and from 18-hydroxycorticosterone nil, implying low processivity. DOPC or cardiolipin, which is exclusively located in the inner mitochondrial membrane, maximized aldosterone yield. In sharp contrast, reconstitution of homologous P450 11B1 with DOPC significantly decreased corticosterone formation without affecting the synthesis of 18-hydroxycorticosterone. The intrinsic fluorescence of P450 17A1 and 11B2 increased in the presence of DOPC, DLPC and PE. Acrylamide quenching studies showed that PE decreased solvent accessibility for tryptophan in P450 17A1, as did 20:4 PC or 18:2 PC for P450 11B2. A moderately positive correlation between the proportion of high-spin substrate-bound species and catalytic activity was only observed in the presence of phosphatidylcholines with low-temperature phase transition. These results demonstrate the potential for phospholipids to regulate the activity of steroidogenic P450 activities and thereby steroid hormone biosynthetic pathways.

Keywords: P450 11B2; P450 17A1; Phospholipid.

Figure 1

Effect of phospholipids on P450 17A1 activities. (A) The 17- and 16α-hydroxylase and 17,20-lyase activities of the purified P450 17A1 as a function of the DLPC/P450 ratio from 50:1 to 1000:1. Incubations were carried out with 10 μM progesterone for 17- and 16α-hydroxylase and 20 μM 17Preg for 17,20-lyase activity, and the products were quantitated by HPLC analysis. All values are means of duplicate experiments. (B) Hydroxylase activities of P450 17A1 in the presence of 6 different PCs, mix of PC:PE at 1:1, or PC:PE:PI at 73.5:24.5:2. Purified P450 17A1 (10 pmol) and POR were reconstituted with P450:POR:phospholipid at a molar ratio of 1:1:100. Incubation was carried out with 10 μM progesterone, and the 17- and 16α-hydroxylated products were quantitated by HPLC analysis. (C) The 17,20-lyase activity of P450 17A1 in the presence of various lipids as in (B). Incubations contained a 1:1:1:100 molar ratio of P450:POR:cytochrome b₅:phospholipid and 20 μM 17Preg, and the product dehydroepiandrosterone was further converted to androstenedione as described in Materials and Methods. Error bars indicate mean ± standard deviation of triplicate assays.

Figure 2

Phospholipid dependence of the intrinsic fluorescence spectra of P450 17A1. (A) Intrinsic fluorescence spectra of P450 17A1 (1 μM) in 50 mM potassium phosphate buffer (pH 7.4) alone and in the presence of PC (100 μM) with various acyl chains. (B) Intrinsic fluorescence spectra of P450 17A1 (1 μM) alone and in the presence of 100 μM PE (18:0/18:1), or PI from bovine liver, which contains 18:0 (46%), 20:4 (17%), and 20:3 (13%). (C) Acrylamide quenching of P450 17A1 intrinsic fluorescence in the absence and presence of various phospholipids. All values are means of duplicate experiments, and data were analyzed using a Stern-Volmer plot. (D) Lehrer plot shows change in tryptophan fluorescence versus the inverse of acrylamide concentration. The intercept on the F_o/ΔF axis gives the inverse of the fraction of accessible tryptophan residues for P450 17A1 in the absence or presence of various phospholipids.

Figure 3

(A–H) The absorption spectra of P450 17A1 in 50 mM potassium phosphate buffer (pH 7.4) in the absence or presence of pregnenolone (Preg) with indicated phospholipid (all at 25 °C). The final concentration of the phospholipid was 100 μM. Spectra are representative of duplicate experiments.

Figure 4

Effect of DOPC on P450 11B2 and P450 11B1 activities. (A) The activity of purified P450 11B2 as a function of the DOPC/P450 molar ratio from 0:1 to 600:1. Incubations were carried out with P450:AdR:Adx at 1:1:60 molar ratios and 200 μM DOC. All values are means of duplicate experiments. (B) The 11β-, 18-hydroxylase and 18-oxidase of P450 11B2 in the presence of 4 different phospholipids when incubated with 200 μM DOC. Results are shown as % products after complete substrate consumption, and error bars indicate mean ± standard deviation of triplicate assays. (C) The 11β-and 18-hydroxylase of P450 11B1 in the presence of 4 different phospholipids when incubated with 200 μM DOC as described in Materials and Methods.

Figure 5

Stimulation of DOPC on P450 11B2 activities. (A) 11β-, 18-hydroxylase and 18-oxidase of P450 11B2 in the presence of 6 different PCs when incubated with 200 μM DOC. (B) 11β-, 18-hydroxylase and 18-oxidase of P450 11B2 in the presence of DLPC, DOPC, CL, PE, mix of PC:CL at 1:1, or PC:PE:CL at 2:2:1 when incubated with 200 μM DOC. Results are shown as % products after complete substrate consumption, and error bars indicate mean ± standard deviation of triplicate assays.

Figure 6

Influence of phospholipid on the intrinsic fluorescence spectra and substrate binding spectra of P450 11B2. (A) Intrinsic fluorescence spectra of P450 11B2 (1 μM) in 50 mM potassium phosphate buffer (pH 7.4) alone and in the presence of 200 μM PC with various acyl chains. (B) Intrinsic fluorescence spectra of P450 11B2 (1 μM) alone and in the presence of 200 μM PE (18:0/18:1), or cardiolipin from bovine heart, which contains 18:2 (90%), and 18:1 (5%). (C) Acrylamide quenching of P450 11B2 intrinsic fluorescence in the absence and presence of various phospholipids. All values are means of duplicate experiments, and data were analyzed using a Stern-Volmer plot. (D) Lehrer plot shows change in tryptophan fluorescence versus the inverse of acrylamide concentration. The intercept on the F_o/ΔF axis gives the inverse of the fraction of accessible tryptophan residues for P450 11B2 in the absence or presence of various phospholipids. (E–H) The absorption spectra of P450 11B2 in 50 mM potassium phosphate buffer (pH 7.4) in the absence or presence of DOC with indicated phospholipids at a final concentration of 200 μM. All spectra were recorded at 25 °C and are representative of duplicate experiments.