Oxidation of 7-dehydrocholesterol and desmosterol by human cytochrome P450 46A1.

Cytochrome P450 (P450 or CYP) 46A1 is expressed in brain and has been characterized by its ability to oxidize cholesterol to 24S-hydroxycholesterol. In addition, the same enzyme is known to further oxidize 24S-hydroxycholesterol to the 24,25- and 24,27-dihydroxy products, as well as to catalyze side-chain oxidations of 7α-hydroxycholesterol and cholestanol. As precursors in the biosynthesis of cholesterol, 7-dehydrocholesterol has not been found to be a substrate of P450 46A1 and desmosterol has not been previously tested. However, 24-hydroxy-7-dehydrocholesterol was recently identified in brain tissues, which prompted us to reexamine this enzyme and its potential substrates. Here we report that P450 46A1 oxidizes 7-dehydrocholesterol to 24-hydroxy-7-dehydrocholesterol and 25-hydroxy-7-dehydrocholesterol, as confirmed by LC-MS and GC-MS. Overall, the catalytic rates of formation increased in the order of 24-hydroxy-7-dehydrocholesterol < 24-hydroxycholesterol < 25-hydroxy-7-dehydrocholesterol from their respective precursors, with a ratio of 1:2.5:5. In the case of desmosterol, epoxidation to 24S,25-epoxycholesterol and 27-hydroxylation was observed, at roughly equal rates. The formation of these oxysterols in the brain may be of relevance in Smith-Lemli-Opitz syndrome, desmosterolosis, and other relevant diseases, as well as in signal transduction by lipids.

and 1.0 mM arabinose. Cultures were incubated for another 42 h at 27°C and 225 rpm in a Multifors incubator, followed by harvesting of cells by centrifugation at 3,500 g for 15 min (Sorvall RC 3B Plus centrifuge, H6000A/HBB6 rotor).

Purifi cation of human P450 46A1
All operations were done at 4°C. The recovered cell pellets (from 6 l of culture) were resuspended in 650 ml of TES buffer [100 mM Tris-acetate (pH 7.5) containing 0.5 M sucrose and 0.5 mM EDTA] per liter of cell culture. Cells were mixed with lysozyme (2 mg/l of cell culture) and kept on ice for 60 min. Spheroplasts were obtained by centrifuging the cell suspension at 3,500 g for 15 min. The resulting spheroplasts were resuspended in 50 ml of sonication buffer [100 mM potassium phosphate (pH 7.5) containing 16% glycerol (v/v), 9 mM magnesium acetate, 100 M dithiothreitol, 1.0 mM phenylmethylsulfonyl fl uoride, and two protease inhibitor tablets (Roche) per liter of cell culture]. After sonication on ice, the material was centrifuged at 12,000 g for 15 min, and the recovered supernatant was further centrifuged at 1.4 × 10 5 g for 60 min. The pellet obtained after ultracentrifugation was collected, homogenized, and solubilized in 600 ml of solubilization buffer [100 mM potassium phosphate buffer (pH 7.5) containing 20% glycerol (v/v), 0.1 mM EDTA, 10 mM ␤ -mercaptoethanol, 0.5 M KCl, and 1.0% sodium cholate (w/v)]. The resulting material was centrifuged at 1.4 × 10 5 g for 60 min, and the supernatant was loaded on a 1.5 × 8 cm Ni +2 -nitrilotriacetic acid column, which had been pre-equilibrated with 100 mM potassium phosphate buffer (pH 7.5) containing 20% glycerol (v/v), 10 mM ␤ -mercaptethanol, 0.5 M KCl, and 1.0% sodium cholate (w/v). The column was washed with the same buffer containing 20 mM imidazole, and P450 46A1 was eluted with the same buffer containing 200 mM imidazole. The eluted fractions containing P450 were dialyzed against 2 l of 100 mM potassium phosphate buffer (pH 7.5) containing 20% glycerol (v/v, 24 h, three buffer changes). The concentration of P450 was measured spectrally ( 26 ) with an OLIS/Aminco DW2 spectrophotometer (On-Line Equipment Systems, Bogart, GA). A typical yield of P450 46A1 from 6 l of culture was 130 nmol after purifi cation.

Expression of NADPH-P450 reductase
The rat enzyme was expressed in E. coli and purifi ed as described elsewhere ( 27 ).

LC-MS
LC-MS was done using a Waters Acquity UPLC system connected to a Thermo LTQ mass spectrometer (see below for LC conditions). Analyte (10 l, otherwise stated) was injected into the system with a partial loop in the needle overfi ll mode. An atmospheric pressure chemical ionization (APCI) + source was used in all cases with the following parameters: discharge voltage, 4.1 kV; discharge current, 5.1 A; vaporizer temperature, 450°C; sheath gas fl ow rate, 50 arbitrary units; auxiliary gas fl ow rate, 5 arbitrary units; sweep gas fl ow rate, 5 arbitrary units; capillary voltage, 18 V; capillary temperature, 275°C; and tube lens voltage, 80 V.

GC-MS
GC-MS analysis was performed using a gas chromatograph (Agilent 6890 GC) coupled with a mass selective detector (Agilent MSD 5973). An Agilent-J and W Scientifi c GC capillary column (DB-5MS; 25 m length, 0.2 mm inside diameter, 0.33 m fi lm thickness) was used. Helium was used as the carrier gas, maintained at a constant fl ow rate of 1.5 ml/min. The GC temperature program was as follows: initial temperature 70°C for 1 min; program from 70 to 130°C at a rate of 50°C/min, hold for 1 min at 130°C; program SLOS and/or cerebrotendinous xanthomatosis ( 12,21 ), providing support for such enzymatic transformation in vivo. Desmosterol, an alternate immediate precursor of cholesterol, contains a double bond at C24 and we hypothesized that, analogous to the reactions of 7-dehydrocholesterol and P450 7A1, desmosterol could be a substrate for P450 46A1, leading to 24,25-epoxycholesterol, 24-ketocholesterol, and/or some side-chain hydroxylated product.
The present work was designed to study the metabolic reactions of 7-dehydrocholesterol and desmosterol by cytochrome P450 46A1. In addition to cholesterol, both 7-dehydrocholesterol and desmosterol were found to be substrates of CYP46A1. Both hydroxylated and epoxidized products (but no ketone) were identifi ed from desmosterol, and only hydroxylated products were identifi ed from 7-dehydrocholesterol by LC-MS, NMR, and GC-MS studies ( Fig. 1 ). The rates of formation of these products were compared with cholesterol oxidation to 24-hydroxycholesterol.

Codon optimization and choice of vector
Codon optimization for P450 46A1 was done using on-line software available through Integrated DNA Technologies (Coralville, IA ). A DNA containing an optimized coding sequence (supplementary Fig. I) and a His 6 tag were synthesized by Genewiz (South Plainfi eld, NJ) and ligated into a pCW vector using NdeI and XbaI restriction sites. The expression vector pCW ( 22 ) has been successfully used for the expression of a number of mammalian P450 enzymes ( 23,24 ), including human P450 46A1 ( 1 ).

Expression of human P450 46A1
A fresh overnight culture was prepared by adding 20 l of a glycerol stock of Escherichia coli (DH5 ␣ ) cells containing the plasmid for the P450 46A1 cDNA, plus a plasmid containing the gene for the E. coli molecular chaperone GroEL/ES, to 100 ml of Luria-Bertani medium containing ampicillin (100 g/ml) and kanamycin (50 g/ml). This starter culture was grown overnight at 37°C and 220 rpm. Large-scale expression of P450 46A1 was done with 6 l of Terrifi c broth medium supplemented with the same antibiotics (see above), trace elements (250 l/l of culture) ( 25 ), 1 mM NaCl, 1 mM thiamine, 2 ml of glycerol per liter of culture, and an overnight starter culture (at a dilution of 1:100, v/v). Cultures were incubated for at least 6 h with shaking at 37°C and 220 rpm, until the optical density at 600 nm reached 0.7. The expression of P450 46A1 was induced by the addition of 1.0 mM isopropyl ␤ -D-1-thiogalactopyranoside, 1.0 mM 5-aminolevulinic acid, patterns of TMS ether derivatives of the products were analyzed for structure elucidation. Derivatization was done using N , Obis (trimethylsilyl)trifl uoroacetamide:dimethylformamide in a 1:1 ratio (v/v) at 23°C for 60 min, and reactions were directly subjected to GC-MS analysis.

Large-scale incubation of P450 46A1 with desmosterol for NMR
NMR was used to identify the structure of the other unknown oxidized product of desmosterol. In order to obtain a suffi cient amount of the products for NMR analysis, a large-scale incubation was performed. P450 46A1 (50 nmol) was incubated with 100 M desmosterol in 50 ml of 100 mM potassium phosphate buffer (pH 7.5) containing 50 nmol of NADPH-P450 reductase and 150 M L-␣ -1,2-dilauroyl-sn -glycero-3-phosphocholine. After initial incubation for 30 min at 37°C, the enzymatic reaction was initiated by the addition of 15 ml of an NADPH-generating system ( 28 ) and incubated for 2 h at 37°C. The reaction was quenched with CH 2 Cl 2 and product was extracted twice with 80 ml of CH 2 Cl 2 , with centrifugation at 2,000 g for 10 min. The organic layers were combined and dried under a nitrogen stream. The dried sample was dissolved in CH 3 CN:CH 3 OH (80:20, v/v) and centrifuged as above to precipitate the suspended particles; the organic layer was collected carefully and subjected to purifi cation. Purifi cation was done with a Waters Acquity UPLC system using an ACQUITY UPLC BEH octadecylsilane (C 18 ) column (1.7 m, 2.1 mm × 100 mm) at 35°C with a fl ow rate of 0.6 ml/min and an injection volume of 15 l. Because of the lack of chromophore in the analyte, LC-MS was used for detection, with an 8:2 (v/v) splitter into the mass spectrometer. Mobile phase A consisted of 95:5 (v/v) water and CH 3 CN with 0.1% HCO 2 H (v/v); mobile phase B consisted of 5:95 (v/v) water and CH 3 CN with 0.1% HCO 2 H (v/v). The LC method was as follows: 0-4.0 min, 80% mobile phase B (v/v); 4.0-4.5 min, 80% mobile phase B (v/v) to 100% mobile phase B; 4.5-8.0 min, 100% mobile phase B; 8.0-8.5 min, 100% mobile phase B to 80% mobile phase B (v/v); 8.5-12.5 min, 80% mobile phase B (v/v). Purifi ed fractions were combined, extracted into CH 2 Cl 2 , dried under nitrogen, and redissolved in CDCl 3 for NMR analysis.

Kinetic assays
Kinetic assays with 7-dehydrocholesterol, desmosterol, and cholesterol were performed as described above but with substrate concentrations varying from 1 M to 100 M {substrates were dissolved in 310 mM HP ␤ CD [45% HP ␤ CD (w/v)], and 5 l of stock substrate solution was added to a 500 l fi nal reaction volume }. The same LC method was used for cholesterol products as in the case of 7-dehydrocholesterol. A product at m/z 385 was detected in the cholesterol assay (24 S -hydroxycholesterol). The k cat and K M values were calculated using the program Dynafi t ( 29 ).

Binding assays
For binding studies, a series of stock solutions of varying concentrations (of each substrate) were prepared in 310 mM HP ␤ CD [45% HP ␤ CD (w/v)]. Binding of various substrates was done in a fi nal volume of 0.33 ml containing 100 mM potassium phosphate (pH 7.5) and 2 M P450 46A1, and substrate was varied from 1-100 M (3.3 l of stock solution of substrate was added). The same volume of cyclodextrin without substrate (3.3 l) was added to a reference cuvette, and spectra were recorded from 350 to 500 nm . The ⌬ A 389 -A 421 , ⌬ A 387 -A 419 , and ⌬ A 389 -A 422 differences for 7-dehydrocholesterol, desmosterol, and cholesterol (respectively) were plotted against increasing concentrations of substrate and binding dissociation constants ( K d ) were estimated by using the program Dynafi t ( 29 ). from 130 to 300°C at a rate of 15°C/min, hold at 300°C for 15 min. Analyte was injected in a splitless mode. MS conditions were as follows: electron impact mode at an ionization energy of 70 eV, transfer line and ion source temperature at 280 and 230°C, respectively. Mass spectra were recorded from m/z 50-700.

NMR
A 600 MHz Bruker NMR spectrometer was used to acquire NMR spectra. The NMR spectrometer was equipped with a 5-mm Z-gradient TCI cryo-probe and Topspin software was used to analyze the data. CDCl 3 was used as a solvent. The peak at ( ␦ ) 7.26 ppm for CHCl 3 was used as a reference in 1 H NMR. 2D NMR was obtained with an optimized 90° pulse width.

Enzymatic assays
Enzymatic assays with 7-dehydrocholesterol and desmosterol were carried out in a 0. , and 2 U/ml yeast glucose 6-phosphate dehydrogenase) ( 28 ) and incubated for 15 min at 37°C. The reaction was quenched with CH 2 Cl 2 (2 ml), and the products were extracted twice with 2 ml of CH 2 Cl 2 and centrifuged at 2,000 g for 10 min (23°C). The organic layers were combined and dried under a nitrogen stream. The dried samples were dissolved in CH 3 CN and subjected to LC-MS analysis. LC-MS was performed using a Waters Acquity UPLC system and ACQUITY UPLC BEH octadecylsilane (C 18

Identifi cation of reaction products
The various products obtained from 7-dehydrocholesterol, desmosterol, and cholesterol assays with P450 46A1 were confi rmed either by comparison with authentic standards, by LC-MS and GC-MS (of TMS ethers), or by LC-MS and NMR. The stereochemistry of 24,25-epoxycholesterol was confi rmed by comparison with standards of 24 R ,25-and 24 S ,25-epoxycholesterol separated on a Waters Alliance (2695) HPLC system that was coupled to a Thermo LTQ mass spectrometer using a chiral column [CHIRALPAK AD-H column; 5 m, 4.6 mm × 250 mm; solvent condition: 5% C 2 H 5 OH in hexanes (v/v); fl ow rate, 1.0 ml/min; temperature, 23°C]. The MS conditions were the same as described above.

Trimethylsilylation of enzymatic products for GC-MS analysis
The oxidative products of 7-dehydrocholesterol were converted into TMS ether derivatives for GC-MS analysis. The fragmentation DNA sequence optimized for E. coli expression (supplementary Fig. I). The purifi ed protein showed a band at 450 nm in the reduced-CO difference spectrum ( Fig. 2 ). The Soret band was observed at 417 nm (data not shown), a characteristic feature of ferric low-spin P450. SDS-PAGE gel showed a single major band at 50 kDa, accounting for >94% of the protein as judged by densitometry of the Coomassie staining ( Fig. 3 ).

Data analysis
Steady-state kinetics and binding studies were fi t using the program Dynafi t ( 29,30 ).
Each substrate was dissolved in 310 mM HP ␤ CD. The fi nal concentration of HP ␤ CD in each reaction mixture was 3.1 mM. The binding constant ( K d ) of cholesterol with HP ␤ CD is reported to be 1.05 mM ( 31 ), and the same value was used in calculations for 7-dehydrocholesterol and desmosterol, in that the calculated logarithms of the octanol-water partition coeffi cients (cLogP values) for cholesterol, 7-dehydrocholesterol, and desmosterol are of the same order of magnitude, i.e., 6.52, 6.30, and 6.04, respectively (ChemDraw, Cambridgesoft, Cambridge, MA). The concentration of free substrate was calculated using the cholesterol K d value of 1.05 mM and a simple binding model (i.e., substrate + HP ␤ CD substrate-HP ␤ CD ). In the cases of 7-dehydrocholesterol and desmosterol, where the affi nity of the steroids for the cyclodextrin would be less due to the lower cLogP values, the effect of using the K d value of 1.05 mM would be to raise the apparent concentration of the sterol and thus overestimate the K M or K d for dissociation of the sterol-P450 46A1 complex.

Protein expression and purifi cation
P450 46A1 was expressed in E. coli cells. We were unsuccessful in the expression of the native sequence and did not obtain good expression in this system until we used a  or desmosterol plus one oxygen atom). The product eluted at t R 6.4 min in the 7-dehydrocholesterol assay was identifi ed as 24-hydroxy-7-dehydrocholesterol by comparison with an authentic standard ( Fig. 4C ). Further, the fragmentation pattern obtained by collision-induced dissociation for 24-hydroxy-7-dehydrocholesterol matched that of the standard (i.e., MS 2 at m/z 383.3 and MS 3 at m/z 365.3, respectively) ( Fig. 6 ). The product 24-hydroxy-7-dehydrocholesterol was further confi rmed by GC-MS (supplementary Fig. II). Similarly, the product at t R 6.4 min from the desmosterol assay was identifi ed as 24,25-epoxycholesterol by comparison with a commercial standard ( Fig. 5B, C ). The stereochemistry of 24,25-epoxycholesterol was confi rmed to be 24 S by comparing it with standards available using a chiral column (supplementary Fig.  III). The fragmentation pattern of the product matched with that of a 24 S ,25-epoxycholesterol standard (data not shown). The desmosterol product ( t R 3.9 min) was not 24-ketocholesterol, as judged by comparison with an authentic standard. The reaction with cholesterol yielded only one product, 24 S -hydroxycholesterol, confi rmed with commercially available standard (supplementary Fig. IV). The identifi cation of the unknown products of 7-dehydrocholesterol and desmosterol is presented below.

Characterization of a second product from 7-dehydrocholesterol
In order to characterize the unknown peak eluting at t R 5.5 min, the collected product was converted to a TMS ether derivative and subjected to GC-MS analysis . GC-MS of desmosterol with P450 46A1 also yielded two products, t R 3.9 and 6.4 min, ( Fig. 5B ). No product was observed in control assays without P450 46A1 ( Figs. 4A, 5A ). All products were observed at m/z 383, which corresponds to dehydration of ions with an m/z of 401 (7-dehydrocholesterol  the nuclear Overhauser effect (NOE) spectroscopy experiment (supplementary Fig. VD), there was a strong NOE between H-24 and a proton in the terminal methylene group, but no NOE between H-24 and the terminal methyl group, indicating the product is 27-hydroxydesmosterol (terminal methylene group is syn to H-24 to produce the NOE correlated spectroscopy signal).

Binding analysis
The calculated K d values for 7-dehydrocholesterol, desmosterol, and cholesterol were 1.4 ± 0.1 M, 1.0 ± 0.4 M, showed two peaks at m/z 544 (M + for TMS ether derivative of a mono-oxygenation product of 7-dehydrocholesterol) (supplementary Fig. II). One peak was identifi ed as 24hydroxy-7-dehydrocholesterol from its fragmentation pattern (supplementary Fig. II), confi rming the results from LC-MS analysis. Analysis of the fragmentation of the second peak led to its assignment as 25-hydroxy-7-dehydrocholesterol ( Fig. 7 ). The assignment is based on the loss of TMS isopropyl ether from the parent ion ( ␣ -cleavage).

Characterization of the second product from desmosterol
The unknown desmosterol product was characterized by NMR. We speculated that this product was a hydroxylated product, formed at an allylic position, in that preliminary analyses had ruled out a C24 ketone (see above). In the 1 H NMR spectrum, a peak from ␦ 5.08 ppm in desmosterol (1H, m, C24) was shifted downfi eld to ␦ 5.38 ppm in the product (1H, m, C24) with similar splitting patterns, suggesting a more electron-defi cient environment of this proton ( Fig. 8 ). On the other hand, a new peak appeared at ␦ 3.99 ppm with an integration of two protons, suggesting that it is a methylene group next to an oxygen atom ( Fig. 8 ; confi rmed by heteronuclear single-quantum correlated spectroscopy, supplementary Fig. VB). On the basis of 1 H NMR ( Fig. 8 ) and homonuclear correlation spectroscopy, heteronuclear multiple-bond correlation spectroscopy, and heteronuclear single-quantum correlation spectroscopy spectra (supplementary Fig. V), the unknown product was characterized as either 27-hydroxydesmosterol or 26-hydroxydesmosterol. In machinery in them. Alternatively, the expression levels of added P450s are relatively low in such cells, and the conversion rate might have been too low for detection. A third possibility is that the oxidation products of 7-dehydrocholesterol might have been further metabolized by other enzymes in the HEK293 cells.
The binding of all substrates showed typical "type I" difference spectra (supplementary Fig. VII). The estimated dissociation constant ( K d ) for 7-dehydrocholesterol was less than that for cholesterol, indicating stronger binding to P450 46A1.The K d for desmosterol was similar to that of 7-dehydrocholesterol, but it was found to be a less effi cient substrate compared with 7-dehydrocholesterol.
Under normal physiological conditions, the cholesterol product 24 S -hydroxycholesterol is the dominant oxysterol in the adult brain (32)(33)(34)(35), modulating cholesterol homeostasis through activation of LXRs ( 3,4 ) and inhibition of the SREBP2 pathway by binding INSIG ( 5 ). A reduced level of 24 S -hydroxycholesterol has been associated with aging and severe neurological diseases ( 36,37 ). A decreased level of 24 S -hydroxycholesterol has also been reported in plasma of SLOS patients ( 13 ), likely due to and 2.2 ± 0.4 M, respectively (corrected for the binding to HP ␤ CD) (supplementary Figs. VII, VIII).

DISCUSSION
This study demonstrates that 7-dehydrocholesterol and desmosterol are substrates of P450 46A1, leading to 24-hydroxy-and 25-hydroxy-7-dehydrocholesterol and to 24 S ,25-epoxycholesterol and 27-hydroxydesmosterol as the products, respectively. The rate of formation ( k cat ) of 24-hydroxy-7-dehydrocholesterol was about one-third that of 24 S -hydroxycholesterol, but the formation of 25-hydroxy-7-dehydrocholesterol was a much more favored reaction ( Table 1 ). The enzyme effi ciency ( k cat / K m ) was similar for 7-dehydrocholesterol compared with cholesterol but 2-3 times higher than that of desmosterol. We note that although previous work by Björkhem et al. ( 13 ) did not fi nd 7-dehydrocholesterol as a substrate of P450 46A1; their work was carried out in HEK293 cells, which presumably would metabolize 7-dehydrocholesterol to cholesterol effi ciently with intact cholesterol biosynthesis observed in the urine of SLOS patients, suggesting that 7-dehydrocholesterol is also a substrate of sterol side-chain cleavage enzyme (P450scc, P450 11A1) ( 41,42 ).
The formation of 24 S ,25-epoxycholesterol directly from desmosterol may have signifi cant biological and physiological consequences. Until now, squalene 2,3;22,23-diepoxide was the only known biosynthetic precursor to 24 S ,25epoxycholesterol ( 43 ), but our study reveals a novel pathway for the formation of this biologically important oxysterol. 24 S ,25-epoxycholesterol was found to be the most potent and abundant oxysterol ligand of LXRs in developing mouse brain (signifi cantly higher than the level of 24 Shydroxycholesterol), and it promotes neurogenesis and neuronal survival in an LXR-dependent fashion ( 44,45 ). Notably, the levels of desmosterol are also high in developing brains (up to 30% of total brain sterols) (46)(47)(48) and defi ciency in cholesterol. We have previously identifi ed 24-hydroxy-7-dehydrocholesterol in the brain tissues of SLOS rodent models ( 12,38 ). However, to what extent the 24-hydroxy-7-dehydrocholesterol could compensate for the lack of 24 S -hydroxycholesterol in terms of its biological functions remains to be elucidated.
It is not entirely unexpected to identify 7-dehydrocholesterol as a substrate of P450. In addition to our fi ndings on this sterol being a substrate of P450 7A1 ( 20 ) and P450 46A1 (current study), it has also been reported that P450 27A1 catalyzes the metabolism of 7-dehydrocholesterol to 25-hydroxy-7-dehydrocholesterol and 26/27-hydroxy-7dehydrocholesterol ( 13,39,40 ). Interestingly, both oxysterols were found to be activators of LXRs, slightly less potent than their cholesterol analogs ( 39,40 ). Furthermore, 7-dehydrocholesterol-derived steroids have been Fig. 7. GC-MS fragmentation pattern of TMS ether product of unknown peak ( t R 5.5 min) in LC-MS profi le of 7-dehydrocholesterol assay ( Fig. 4B ). The fragment at m/z 413 confi rmed the product to be 25-hydroxy-7-dehydrocholesterol. been shown in a mouse model ( 53 ). We have not examined the effects of any of these drugs on the new reactions we characterized here.
In conclusion, we have identifi ed 7-dehydrocholesterol and desmosterol as substrates of P450 46A1. The oxysterols derived from these enzymatic pathways could have signifi cant biological consequences in related physiological or pathological conditions where the levels of these two cholesterol precursors are high, such as in SLOS (7-dehydrocholesterol) ( 7,8 ), desmosterolosis (desmosterol) ( 7,54 ), and developing brain (desmosterol) (46)(47)(48). The direct conversion of desmosterol to 24 S ,25-epoxycholesterol by P450 46A1 represents a distinctly new pathway for the formation of this potent oxysterol ligand of LXRs ( 4,44,45 ). low in the adult brain ( ‫ف‬ 0.1% of cholesterol) ( 49 ). Thus, our fi nding suggests a possible link between desmosterol and the production of 24 S ,25-epoxycholesterol during brain development. Interestingly, Griffi ths and coworkers recently reported that the level of 24 S ,25-epoxycholesterol was greatly decreased in the brain of Cyp46a1 -knockout mice (one-fi fth of the level in wild-type mice), providing support for the participation of P450 46A1 in the production of this oxysterol in vivo ( 50 ). It is of additional interest that 24 S ,25-epoxycholesterol was found to be an inhibitor of 3 ␤ -hydroxysterol-⌬ 24 -reductase (DHCR24), leading to the accumulation of desmosterol in a number of cell lines ( 51 ).
The substrate selectivity of P450 46A1 is not as limited as once thought. Cholesterol 24 S -hydroxylation has been the main reaction ascribed to P450 46A1. However, several other sterols are also substrates, including 24 S -hydroxycholesterol (25-and 27-hydroxylations), 7 ␣ -hydroxycholesterol, cholestanol, progesterone, and testosterone ( 1 ). Some drugs are also substrates ( 1 ). Here we have shown that recombinant human P450 46A1 catalyzes the 24-and 25-hydroxylation of 7-dehydrocholesterol and the epoxidation and hydroxylation of desmosterol. Interestingly, P450 46A1 binds and oxidizes a number of drugs ( 1,52 ). This is an unusual phenomenon in that most of the P450s that appear to be specialized for oxidation of endogenous substrates do not use xenobiotic chemicals as substrates. The overall in vivo contribution of P450 46A1 to the metabolism of these drugs, even in brain, is unknown. Furthermore, P450 46A1 activity (toward cholesterol) is stimulated by binding to some drugs (e.g., efavirenz, acetaminophen, mirtazapine, galantamine), and the in vivo relevance of this effect has