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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2004-0966
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 2 1210-1219
Copyright © 2005 by The Endocrine Society

CYP3A4 Is a Vitamin D-24- and 25-Hydroxylase: Analysis of Structure Function by Site-Directed Mutagenesis

Ram P. Gupta, You Ai He, Kennerly S. Patrick, James R. Halpert and Norman H. Bell

Departments of Medicine (R.P.G., N.H.B.) and Pharmaceutical Sciences (K.S.P.), Medical University of South Carolina, Charleston, South Carolina 29425; and Department of Pharmacology and Toxicology (Y.A.H., J.R.H.), University of Texas Medical Branch, Galveston, Texas 77555-1031

Address all correspondence and requests for reprints to: Norman H. Bell, M.D., Department of Medicine, Medical University of South Carolina, Strom Thurmond Research Building, 114 Doughty Street, P.O. Box 250775, Charleston, South Carolina 29425. E-mail: belln{at}musc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Studies were performed to identify the microsomal enzyme that 24-hydroxylates vitamin D, whether 25-hydroxylation occurs, and structure function of the enzyme. Sixteen hepatic recombinant microsomal cytochrome P450 enzymes expressed in baculovirus-infected insect cells were screened for 24-hydroxylase activity. CYP3A4, a vitamin D-25-hydroxylase, and CYP1A1 had the highest 24-hydroxylase activity with 1{alpha}-hydroxyvitamin D2 (1{alpha}OHD2) as substrate. The ratio of rates of 24-hydroxylation of 1{alpha}-hydroxyvitamin D3 (1{alpha}OHD3), 1{alpha}OHD2, and vitamin D2 by CYP3A4 was 3.6/2.8/1.0. Structures of 24-hydroxyvitamin D2, 1,24(S)-dihydroxyvitamin D2, and 1,24-dihydroxyvitamin D3 were confirmed by HPLC and gas chromatography retention time and mass spectroscopy. In characterized human liver microsomes, 24-hydroxylation of 1{alpha}OHD2 by CYP3A4 correlated significantly with 6ß-hydroxylation of testosterone, a marker of CYP3A4 activity. 24-Hydroxylase activity in recombinant CYP3A4 and pooled human liver microsomes showed dose-dependent inhibition by ketoconazole, troleandomycin, {alpha}-naphthoflavone, and isoniazid, known inhibitors of CYP3A4. Rates of 24- and 25-hydroxylation of 1{alpha}OHD2 and 1{alpha}OHD3 were determined in recombinant wild-type CYP3A4 and site-directed mutants and naturally occurring variants expressed in Escherichia coli. Substitution of residues showed the most prominent alterations of function at residues 119, 120, 301, 305, and 479. Thus, CYP3A4 is both a 24- and 25-hydroxylase for vitamin D2, 1{alpha}OHD2, and 1{alpha}OHD3.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
VITAMIN D AND related compounds are widely used for the treatment of a host of bone diseases, including rickets, osteomalacia, hypoparathyroidism, pseudohypoparathyroidism, and renal osteodystrophy (1, 2, 3). These include vitamin D2, vitamin D3, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], 1{alpha}-hydroxyvitamin D3 (1{alpha}OHD3), and 1{alpha}-hydroxyvitamin D2 (1{alpha}OHD2) (1, 2, 3). In addition to 1,25-dihydroxyvitamin D2 [1,25-(OH)2D2], 1,24(S)-dihydroxyvitamin D2 [1,24(S)-(OH)2D2] is another metabolite of 1{alpha}OHD2 (4, 5, 6). Little is known about the enzymes that 24-hydroxylate vitamin D and its analogs, their identity or their structure function. 24-Hydroxyvitamin D2 (24OHD2) is produced by Hep3B cells (5). Recombinant CYP27A1, a mitochondrial enzyme involved in the alternative pathway of bile acid metabolism, also produces 24OHD2 and 25-hydroxylates vitamin D3 but not vitamin D2 (7). Homology does not necessarily predict function. CYP2D6 protein, a human hepatic microsomal enzyme involved in drug metabolism that has 77% identity with CYP2D25 protein, a porcine hepatic enzyme that 25-hydroxylates vitamin D2 and vitamin D3, does not 25-hydroxylate either vitamin D2 or vitamin D3 (8). In the present study, major hepatic microsomal cytochrome P450 enzymes, expressed in baculovirus-infected cells, were screened to identify vitamin D-24-hydroxylase activity, an approach previously used to identify CYP3A4 as a vitamin D-25-hydroxylase (9). We identified the hepatic microsomal enzyme that 24-hydroxylates vitamin D2 and its analogs, confirmed identity of the metabolites, and studied structure-function relationship of 24- and 25-hydroxylation of the enzyme by site-directed mutagenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Microsomes of human recombinant cytochrome P450 from liver coexpressed in baculovirus-infected insect cells with human cytochrome P450 reductase and, in some cases, cytochrome b5, individual human liver microsomes with characterized enzyme activity, pooled human liver microsomes, and a reduced nicotinamide adenine dinucleotide phosphate (NADPH)-regenerating system, were purchased from BD Gentest (Woburn, MA). The recombinant CYP3A4 expressed in baculovirus-infected cells contained 1 nmol/ml cytochrome P450 enzyme, 11.1 ± 1.2 nmol/ml NADPH cytochrome P450 reductase, and 7.8 ± 0.9 nmol/ml cytochrome b5 (ratio 1/11/8). Wild-type CYP3A4 and its mutants used in structure-function studies were prepared and expressed in Escherichia coli. Troleandomycin, isoniazid, ketoconazole, {alpha}-naphthoflavone, vitamin D2, 1,25-(OH)2D3, NADPH, 3-[(3-cholamidopropyl)di-methylammonio]-1-propanesulfonate (CHAPS), diphenylethane-1,2-diol (DPED), and 1,2-dioleoyl-sn-glycero-3-phosphacholine (DOPC), were purchased from Sigma Aldrich (St. Louis, MO); acetonitrile, dichloromethane, hexanes, methanol, and 2-propanol from Fisher Scientific (Norcross, GA); HEPES from Calbiochem Corp. (La Jolla, CA); and [3H-26,27-methyl]-1,25-(OH)2D3 and [3H-26,27-methyl]-25OHD3 from Amersham Biosciences (Piscataway, NJ). Cytochrome b5 was coexpressed with all of the enzymes except CYP1A1, CYP1A2, CYP1B1, CYP2C18, CYP3A5, and CYP4A11. 1{alpha}OHD2 and 1,24(S)-(OH)2D2 were generously provided by Bone Care International (Madison, WI) and 1,24-dihydroxyvitamin D3 [1,24-(OH)2D3] and 1{alpha}OHD3 by Leo Pharmaceutical Products, Ltd. (Ballerup, Denmark). 24OHD2 was kindly provided by Dr. Ronald Horst (Ames, IA). Characterized liver microsomes had been assayed by the vendor for enzymetic activities of coumarin 7-dehydroxylase (CYP2A6), (S)-mephenytoin N-demethylase (CYP2B6), paclitaxel 6{alpha}-hydroxylase (CYP2C8), diclofenac 4'-hydroxylase (CYP2C9*1), (S)-mephenytoin 4'-hydroxylase (CYP2C19), testosterone 6ß-hydroxylase (CYP3A4), and lauric acid {omega}-hydroxylase (CYP4A11) as described (10). For studies with CYP3A4 and mutants, NADPH-cytochrome P450 reductase and cytochrome b5 were obtained as described previously (11).

Assay of vitamin D-24- and 25-hydroxylase activities

A 1-ml reaction mixture contained either 50 µl hepatic microsomes (50–150 µg) or 50 µl of microsomes from baculovirus-infected insect cells and 100 µl 0.5-M Na2HPO4 (pH 7.4), 50 µl 60-mM EDTA, 50 µl NADPH regenerating solution A (BD Gentest), 10 µl regenerating solution B (BD Gentest), 2 µl 5-mM dianilinoethane, and 2 µl 1{alpha}OHD2, 1{alpha}OHD3, or vitamin D2. The final concentration of substrate was 10 µM. Unless otherwise indicated, the reaction was performed at 37 C for 1.5 h and terminated with the addition of 1 ml acetonitrile. Protein was measured by the bicinchoninic acid method with a kit (Pierce, Rockford, IL) (12).

Construction, expression, and purification of CYP3A4 and mutants

Plasmid pE3A4His was used to express CYP3A4. Details of construction of CYP3A4 mutants (13, 14, 15) and of naturally occurring CYP3A4 protein variants (16) were as previously described. All constructs were transformed into Escherichia coli strain DH5a or Topp3 (Stratagene, La Jolla, CA). Transformed cells were grown overnight on a Luria Bertani plate containing 50 µg/ml ampicillin at 37 C. A single colony was inoculated in 20 ml Luria Bertani broth with 50 µg/ml ampicillin. After overnight growth at 37 C with vigorous shaking, 15 ml culture was transferred into a 1-liter flask containing 250 ml tryptone broth with 100 µg/ml ampicillin. Cultures were grown at 37 C with shaking at 250 rpm. When cultures had an absorbance of 1.2 at 550 nm, 1 mM isopropylthio-ß-D-galactoside and 80 mg/liter alanine were added. Cells were harvested after an additional 72-h incubation at 30 C with shaking at 190 rpm. CHAPS-solubilized membrane preparations were made as described previously (17). HIS-tagged proteins were purified by column chromatography with TALON metal affinity resin (CLONTECH, Palo Alto, CA) (16). P450 content was determined by carbon monoxide difference spectra in the presence of 1% Triton X-100 added to the protein sample before dilution with microsome-solubilization buffer containing 100 mM potassium phosphate (pH 7.3), 20% glycerol, 0.5% sodium cholate, 0.4% Renex, and 1.0 mM EDTA.

Assay of hydroxylase activities of recombinant CYP3A4 and CYP3A4 mutants

The reconstitution of bacterial-expressed cytochrome P450 enzymes was performed at room temperature in the presence of DOPC (0.1 mg/ml), dithiothreitol (1 mM), CHAPS (0.4%), cytochrome P450 (6 pmol), NADPH cytochrome P450 reductase (24 pmol), and cytochrome b5 (12 pmol) (ratio 1/4/2) in a total vol of 5 µl. The reagents were added in the given order. The solution was diluted to 50 µl that then contained 3[N-morholino]propanesulfonic acid (50 mM), glycerol (10%), and EDTA (1 mM). The reconstituted cytochrome P450 was kept at –80 C until used. The reaction mixture contained reconstituted P450 (50 µl), phosphate buffer (50 mM) (pH 7.4), polyvinyl alcohol (0.3%), EDTA (3 mM), NADPH-regenerating solution A (50 µl), NADPH regenerating solution B (10 µl), DPED (10 µM), and 1{alpha}OHD2, 1{alpha}OHD3, or vitamin D2 (10 µM) in a total vol of 1 ml. The reaction was performed at 37 C for 1.5 h and terminated with the addition of 1 ml acetonitrile.

Extraction of samples

The acetonitrile mixture was mixed with a vortex, 1,000 cpm [3H]-1,25(OH)2D3 or [3H]-25OHD3 was added for recovery, and the sample was then centrifuged at 4 C and 2,000 x g for 15 min (18). The supernatant was decanted to another 13 x 100 mm glass tube containing 1 ml 0.4-M K2HPO4 (pH 10.4, pH adjusted with KOH) and mixed. The solution containing dihydroxylated metabolites was mixed and transferred to a C18OH Cartridge (DiaSorin, Stillwater, MN) that had been conditioned twice with 1.5 ml methanol. The cartridge was washed with 5 ml solvent A (methanol:water, 70:30), 5 ml solvent B (hexanes:dichloromethane, 88:12), 3 ml solvent C (hexanes:2-propanol, 99:1), and eluted with 5 ml solvent D (hexanes:2-propanol, 95:5) (18). The cartridges were washed with 1 ml 2-propanol and conditioned with methanol, as above, for further use. The eluted extracts were evaporated under N2; the residue was dissolved in 200 µl dichloromethane:hexanes:2-propanol, 50:50:2.5, and subjected to HPLC. The supernatant containing the monohydroxylated metabolites was mixed with 0.5 M K2HPO4 (pH 10.4) and transferred, as described above, onto a regenerated C18 cartridge (DiaSorin). The columns were washed with 5 ml solvent A (methanol:water, 70:30) and eluted with 3.5 ml acetonitrile. The solution was evaporated under N2, dissolved in 200 µl hexanes:methylene chloride:2-propanol (50:50:2.5), and subjected to HPLC.

Isolation and measurement of vitamin D metabolites

Extracts in 200 µl solution were loaded onto a Zorbax Sil 4.6 x 250 mm column, and the metabolites were separated by HPLC with hexanes:2-propanol (85:15). 1,24(S)-(OH)2D2, 1,24-(OH)2D3, 1,25-(OH)2D2, and 1,25-(OH)2D3 were quantified by measuring the area of the separated peaks that eluted at 7.2, 7.5, 8.3, and 8.8 min, respectively. Recovery was assessed with [3H]-1,25(OH)2D3. 24OHD2 and 25-hydroxyvitamin D2 (25OHD2) also were quantified by measuring the area of the separated peaks that eluted at 6.9 and 7.9 min, respectively. The lower limit of detection for vitamin D metabolites, by the HPLC method, is 500 pg. 24OHD2 in HPLC eluates was quantified by RIA (19). Recovery was assessed with [3H]-25OHD3. Mean recoveries of [3H]-1,25-(OH)2D3 and [3H]-25OHD3 were 58 ± 2% and 60 ± 2%, respectively. Except where noted, results are expressed as nmol/nmol·1.5 h for human recombinant cytochrome P450 enzymes expressed in baculovirus-infected insect cells and nmol/mg protein·1.5 h for hepatic microsomes. Results of structure-function analysis are expressed as pmol/nmol·1.5 h. All measurements were carried out in triplicate.

Mass spectrometry of 1,24(S)-(OH)2D2, 1,24-(OH)2D3, and 24OHD2

The structural characterization of the metabolites was carried out by liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-MS (GC-MS). The LC-MS (API QStar Pulsar, PE Sciex Instruments, Boston, MA) provided molecular ions consistent with these hydroxylated species. The spray voltage was 800 V, and the collision energy was 25 V. N2 was the collision gas (20). Structural confirmation of 24OHD2, 1,24(S)-(OH)2D2, and 1,24-(OH)2D3 was performed by GC-MS (Agilent 6890 GC-5973N MS with Chemstation and Autosampler, Wilmington, DE). A fraction containing a metabolite from the HPLC separation or a reference standard in ethanol was evaporated to dryness in a tapered microvial insert (200 µl; spring fitted) with a stream of N2. N,O-bis(trimethylsilyl)trifluoroacetamide (Supelco, Bellefonte, PA, 25 µl) was added, then the microvial was sealed in a 1.5-ml Autosampler vial with a Teflon lined cap. After heating for approximately 24 h at 42 C, 1–2 µl was injected in the pulsed splitless mode onto a 5% phenylmethylpolysiloxane GC column (30 m x 0.25 mm; film, 0.25 µm) with the helium linear velocity at 55 cm/sec. The oven was held at 200 C for 1.5 min, followed by a 20 C/min ramp to a hold at 280 C. Under these GC conditions, the tristrimethylsilyl (TMS) derivative of metabolite or standard of 1,24(S)-(OH)2D2 eluted at 16.7 min, and the corresponding derivatives of 1,24-(OH)2D3 eluted at 15.7 min after injection. MS ionization was by electron impact at 70 eV, acquiring m/z 50–700. The TMS derivative of metabolite and standard of 24OHD2 eluted at 15.5 min after injection.

Statistics

Linear regression analysis was used to correlate hepatic microsomal vitamin D-24-hydroxylase activity with known activities of cytochrome P450 enzymes in characterized liver microsomes.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Fourteen of 16 major hepatic cytochrome P450s expressed in baculovirus-infected insect cells showed 24-hydroxylase activity with 1{alpha}OHD2 and 1{alpha}OHD3 as substrates. CYP3A4 had the highest activity with 1{alpha}OHD3 and CYP3A4, and CYP1A1 had the highest activity with 1{alpha}OHD2 (Fig. 1Go). The rates of 24-hydroxylation of 1{alpha}OHD2 by CYP3A4 and CYP1A1 were the same. The rate of 24-hydroxylation of 1{alpha}OHD3 by CYP3A4 was 1.5-fold higher than that of 1{alpha}OHD2 and 7-fold higher than that of any of the other cytochrome P450s. Eluates from HPLC showed two peaks, an earlier one for 24-hydroxylated products and a later one for 25-hydroxylated products (Fig. 2Go, A–D). Structures of TMS derivatives of 1,25(OH)2D2 and 1,25(OH)2D3 were confirmed in a previous study by comparison of their respective HPLC and GC retention times, molecular ions (M+; LC-MS and GC-MS), and electron impact fragmentation patterns with those of the respective reference standards (9). Structures of TMS derivatives of 1,24(S)-(OH)2D2 and 1,24-(OH)2D3 (Fig. 3Go, A–D) were confirmed in the same manner. Reported literature electron impact MS spectra of TMS derivatives of 1,24(S)-(OH)2D2 and 1,24-(OH)2D3 (5, 6, 7) also were consistent with those of the present study. The {alpha}-cleavage fragmentation process characteristic of electron impact ionization further established hydroxylation at the 24 position. Unlike the 25- or 26(27)-hydroxyl isomers of vitamin D2 and D3 (7, 9), such bond scission of TMS derivatives of 24-OH metabolites of vitamin D2, loss of only an isopropyl radical, yielded the predicted high mass ions m/z 601 and 589, respectively. Molecular ions (M+) were detectable for each of the electron impact MS samples in Fig. 3Go, m/z 644 for 1,24(S)-(OH)2D2 and m/z 632 for 1,24-(OH)2D3. The GC retention time of 16.7 min for metabolically formed TMS derivatives of 1,24(S)-(OH)2D2 and authentic 1,24(S)-(OH)2D2 were identical. It should be noted that the retention times of 1,24(S)-(OH)2D2 and 1,24(R)-(OH)2D2 are different (5). Eluates from HPLC with vitamin D2 as substrate showed two peaks, 24OHD2 and 25OHD2 (Fig. 4Go). The structure of the TMS derivative of 24OHD2 was confirmed by HPLC, and GC retention time of 15.5 min and election impact fragmentation pattern (Fig. 5AGo and 5BGo). Taken together, the identical electron impact MS fragmentation patterns and GC retention times for these TMS derivatives of metabolites, when compared with their respective reference standards, demonstrates that no geometric isomerism had occurred about the alkene bonds.



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  		FIG. 1. Vitamin D-24-hydroxylase [24(OH)ase] activity in microsomes of recombinant human cytochrome P450 enzymes expressed in baculovirus-infected insects. Substrate concentration of 1{alpha}OHD2 and 1{alpha}OHD3 was 10 µM. Incubations were carried out with enzymes for 1.5 h. At the end of incubation, samples were extracted, separated by HPLC, and analyzed by measuring the peaks of 1,24-(OH)2D2 or 1,24-(OH)2D3 in the eluates. Results are mean ± SE of three determinations. To convert from metric to SI units, multiply picomoles by 429 for 1,24-(OH)2D2.

 


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  		FIG. 2. HPLC elution profile of products of incubations of CYP3A4 and human liver microsomes with 1{alpha}OHD2 and 1{alpha}OHD3 as substrates. With 1{alpha}OHD2 as the substrate, 1,24-(OH)2D2 and 1,25-(OH)2D2 were produced and eluted as peaks 1 and 2, respectively, when incubated with recombinant CYP3A4 expressed in baculovirus-infected insect cells (A) and pooled human liver microsomes (B). With 1{alpha}OHD3 as the substrate, 1,24(OH)2D3 and 1,25(OH)2D3 were produced and eluted as peaks 1 and 2, respectively, when incubated with recombinant CYP3A4 (C) and pooled human liver microsomes (D). The large peak before peak 1 is the substrate, either 1{alpha}OHD2 or 1{alpha}OHD3. Substrate concentration was 10 µM. Incubations were carried out for 1.5 h. AU, Arbitrary units.

 


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  		FIG. 3. Electron impact mass spectra of metabolically formed 1,24-(OH)2D2 (A), reference standard of 1,24-(OH)2D2 (B), metabolically formed 1,24-(OH)2D3 (C), and reference standard of 1,24-(OH)2D3 (D). These samples were analyzed as the trimethylsilyl derivatives.

 


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  		FIG. 4. HPLC elution profile of products of incubations of CYP3A4 and human liver microsomes with vitamin D2 as substrate. With vitamin D2 as the substrate, 24OHD2 and 25OHD2 were produced and eluted as peaks 1 and 2, respectively, when incubated with recombinant CYP3A4 expressed in baculovirus-infected insect cells (A) and pooled human liver microsomes (B). The large peak before peak 1 is the substrate vitamin D2. The other large peaks at the beginning are the solvent front and impurities that have not been identified. Substrate concentration was 10 µM. Incubations were carried out for 1.5 h.

 


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  		FIG. 5. Electron impact mass spectra of metabolically formed 24OHD2 (A) and reference standard of 24OHD2 (B). These samples were analyzed as trimethylsilyl derivatives.

 
Studies with recombinant CYP3A4 expressed in baculovirus-infected insect cells and 1{alpha}OHD2 as substrate showed that, whereas 24-hydroxylation varied directly with substrate concentration, 25-hydroxylation reached a peak at 30 µM and did not increase at higher concentrations (Table 1Go). As a consequence, the ratio of 25-hydroxylation to 24-hydroxylation changed by 26-fold when the substrate concentration was increased 200-fold. Studies with recombinant CYP3A4 and 1{alpha}OHD3 as the substrate showed that 25-hydroxylation reached a peak at 10 µM, and 24-hydroxylation reached a peak at 20 µM (Table 2Go), so that ratio decreased very modestly. Studies with the two substrates were performed under identical conditions. In seven experiments with recombinant CYP3A4 and 1{alpha}OHD2, mean 24-hydroxylation was 2,920 ± 216 pmol/nmol·1.5 h, and mean 25-hydroxylation was 12,723 ± 768 pmol/nmol·1.5 h. In seven experiments with recombinant CYP3A4 and 1{alpha}OHD3, mean 24-hydroxylation was 3,720 ± 289 pmol/nmol·1.5 h, and mean 25-hydroxyation was 3,023 ± 206 pmol/nmol·1.5 h. In a single experiment with recombinant CYP3A4 and pooled human liver microsomes, 25-hydroxylation of vitamin D2 was 2,135 ± 126 pmol/nmol·1.5 h (n = 5) and 149 ± 11 pmol/mg protein·1.5 h (n = 5), respectively. In one experiment with recombinant CYP3A4, 24-hydroxylation of vitamin D2 was 1,031 ± 133 pmol/nmol·1.5 h. The ratio of 24-hydroxylation of 1{alpha}OHD3, 1{alpha}OHD2, and vitamin D2 by recombinant CYP3A4 was 3.6/2.8/1.0, whereas the ratio of 25-hydroxylation of 1{alpha}OHD3, 1{alpha}OHD2, and vitamin D2 by recombinant CYP3A4 was 1.4/6.0/1.0.


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  		TABLE 1. Effects of substrate concentration on 24- and 25-hydroxylation of 1{alpha}OHD2 by microsomes of recombinant CYP3A4 expressed in baculovirus-infected cells

 

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  		TABLE 2. Effects of substrate concentration on 24- and 25-hydroxylation of 1{alpha}OHD3 by microsomes of recombinant CYP3A4 expressed in baculovirus-infected cells

 
When two different substrates are metabolized by the same enzyme, enzymatic activity of the two substrates should correlate in samples from different livers. In 12 characterized human liver microsomes in which individual enzyme activities already had been determined (21), CYP3A4 24-hydroxylase activity correlated significantly with testosterone 6ß-hydroxylase activity, a marker of CYP3A4 (Fig. 6Go). In these different liver microsomes, CYP3A4 24-hydroxylase activity varied 12-fold and testosterone 6ß-hydroxylase activity 28-fold. In nine experiments with pooled human liver microsomes, mean 24-hydroxylation of 1{alpha}OHD2 was 112 ± 10 pmol/mg protein·1.5 h.



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  		FIG. 6. Correlation between vitamin D-24-hydroxylase [24(OH)ase] activity and CYP3A4 testosterone 6ß-hydroxylase activity in a panel of 12 characterized human liver microsomes. Incubations were carried out with microsomes for 1.5 h. Substrate concentration of 1{alpha}(OH)D2 was 10 µM. At the end of incubation, samples were extracted, separated by HPLC, and analyzed by measuring the peaks of 1,24-(OH)2D2 in the eluates. Results are mean ± SE of three determinations. To convert from metric to SI units, multiply picomoles by 429 for 1,24-(OH)2D2 and by 304 for 6ß-testosterone.

 
To further investigate whether 24-hydroxylation of 1{alpha}OHD2 in human liver microsomes is mediated by CYP3A4, the effects of known inhibitors of CYP3A4 enzymes on activity in pooled liver microsomes and recombinant CYP3A4 were compared (Fig. 7Go). 24-Hydroxylase activity was inhibited in a dose-response fashion by ketoconazole, troleandomycin, and isoniazid, known inhibitors of CYP3A enzymes (22, 23, 24, 25), and by {alpha}-naphthoflavone, an activator/inhibitor of CYP3A4 (25). 24-Hydroxylase activity of CYP1A1 and pooled liver microsomes was reduced in a dose-response fashion by {alpha}-naphthoflavone (Fig. 8Go). In each case, however, reduction of activity in response to the drug was less with pooled human liver microsomes than with the recombinant enzyme. These findings indicate that additional vitamin D-24-hydroxylases may be present in liver.



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  		FIG. 7. Effects of ketoconazole, {alpha}-naphthoflavone, troleandomycin, and isoniazid on vitamin D-24-hydroxylase activity in pooled human liver microsomes (HLM) and recombinant CYP3A4 expressed in baculovirus-infected insect cells. Substrate concentration of 1{alpha}OHD2 was 10 µM. Incubations were carried out with microsomes or enzymes for 1.5 h. At the end of incubation, samples were extracted, separated by HPLC, and analyzed by measuring the peaks of 1,24-(OH)2D2 in the eluates. Results are mean ± SE of three determinations.

 


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  		FIG. 8. Effects of {alpha}-naphthoflavone on vitamin D-24-hydroxylase activity in pooled HLM and recombinant CYP1A1 expressed in baculovirus-infected insect cells. Substrate concentration of 1{alpha}OHD2 was 10 µM. Incubations were carried out with microsomes or enzymes for 1.5 h. At the end of incubation, samples were extracted, separated by HPLC, and analyzed by measuring the peaks of 1,24-(OH)2D2 in the eluates. Results are mean ± SE of three determinations.

 
We then performed a structure-function analysis of 24- and 25-hydroxylation by reconstituted wild-type CYP3A4 and its mutants expressed in Escherichia coli with 1{alpha}OHD2 and 1{alpha}OHD3 as substrates (Tables 3–5GoGoGo). In three experiments with reconstituted wild-type CYP3A4 and 1{alpha}OHD2, mean 24-hydroxylation was 1001 ± 58 pmol/nmol·1.5 h, and mean 25-hydroxyation was 2892 ± 283 pmol/nmol·1.5 h. In three experiments with reconstituted wild-type CYP3A4 and 1{alpha}OHD3, mean 24-hydroxylation was 423 ± 21 pmol/nmol·1.5 h, and mean 25-hydroxylation was 352 ± 47 pmol/nmol·1.5 h. Thus, enzyme activity of wild-type reconstituted CYP3A4 expressed in Escherichia coli was consistently lower than activity of the recombinant enzyme expressed in baculovirus-infected insect cells. Substitutions of residues in substrate recognition sites 1, 2, 4, 5, and 6 were carried out by site-directed mutagenesis (13, 14, 15, 16, 17). Alterations in residues were associated with 2-fold or greater changes at a number of sites. With 1{alpha}OHD2, increases in 25-hydroxylation were exhibited by S119A, S119F, and L479F and decreases by I301A and A305F (Table 3Go). Increases in 24-hydroxylation were exhibited by S119A, S119F, L210A, I301A, and I301F and decreases by L211A and F213W. With 1{alpha}OHD3, increases in 25-hydroxylation were exhibited by S119A, S119F, I120W, I301F, L479A, and L479F and decreases by I301A and A305F (Table 4Go). Increases in 24-hydroxylation were exhibited by S119A, S119F, I301F, and L479F and a decrease by A305F. Activity of four naturally occurring protein variants of CYP3A4 (G56D, V170I, D174H, and M445T) was examined (16). Of these, a 3-fold increase in 25-hydroxylation was exhibited by M445T with 1{alpha}OHD3 (Table 5Go). None of the other sites were associated with as much as a 2-fold change in activity, compared with the wild-type.


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  		TABLE 3. Structure-function analysis of hydroxylation with 1{alpha}(OH)D2 as substrate by wild-type and mutant CYP3A4 expressed in E. coli

 

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  		TABLE 4. Structure-function analysis of hydroxylation with 1{alpha}OHD3 as substrate by wild-type and mutant CYP3A4 expressed in E. coli

 

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  		TABLE 5. Structure-function analysis of hydroxylation by wild-type and naturally occurring mutants of CYP3A4 expressed in E. coli

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results indicate that CYP3A4 is a hepatic microsomal vitamin D-24-hydroxylase, as well as a vitamin D-25-hydroxylase (9), and provide evidence that CYP3A4 may account for some, if not most, of the 24-hydroxylase activity in liver microsomes. First, CYP3A4 24-hydroxylated 1{alpha}OHD2, 1{alpha}OHD3, and vitamin D2. Along with CYP1A1, CYP3A4 had the highest vitamin D-24-hydroxylase activity of the 16 cytochrome P450 enzymes examined. CYP3A4 is the most abundant cytochrome P450 enzyme expressed in liver, whereas CYP1A1 is poorly expressed (26, 27, 28, 29, 30, 31). Second, there was a highly significant correlation of 24-hydroxylase activity of 1{alpha}OHD2 with CYP3A4-mediated testosterone 6ß-hydroxylase activity in a panel of 12 characterized human liver microsomes (21). The liver microsomes with the highest testosterone 6ß-hydroxylase activity had the highest 24-hydroxylase activity. Third, dose-response studies showed that 24-hydroxylase activity in hepatic microsomes and recombinant CYP3A4 was inhibited in parallel by ketoconazole, {alpha}-naphthoflavone, isoniazid, and troleandomycin, known inhibitors of CYP3A enzymes (22, 23, 24, 25). CYP1A1 may have accounted for some of the activity because 24-hydroxylase activity in recombinant CYP1A1 and pooled liver microsomes was inhibited by {alpha}-naphthoflavone, also an inhibitor of CYP1A1 (26). Again, CYP1A1 is expressed in very low abundance in liver, representing less than 1% of the total CYP450 protein (26, 27, 28, 29, 30, 31). As already mentioned, CYP27A1 also 24-hydroxylates vitamin D2 (7).

In addition to liver, CYP3A4 is expressed in the small intestine, esophagus, colon, kidney, and leukocytes (27, 28, 29, 30, 31, 32, 33). Hepatic expression of CYP3A4 varies by as much as 40-fold (27, 34), and testosterone 6ß-hydroxylase activity in hepatic microsomes by as much as 31-fold (35). In the present study, activity of testosterone 6ß-hydroxylase activity varied by 28-fold. The 16 microsomal enzymes studied are the major drug-metabolizing cytochrome P450 enzymes in human liver, and CYP3A4 is the most abundant (31, 33, 34).

With increasing concentration of 1{alpha}OHD2 as substrate, 25-hydroxylation showed a maximum velocity at 30 µM, whereas 24-hydroxylation showed a continuous increase in velocity (Table 1Go). These findings suggest a change in conformation of the binding site with increasing concentration of substrate. In contrast, with 1{alpha}OHD3 as substrate, both 24- and 25-hydroxylation showed a maximum velocity at 20 µM and 10 µM substrate concentration, respectively, indicating no difference in their kinetics. Formation of different metabolites with the same substrate was found with other substrates of CYP3A4, including midazolam, testosterone, and progesterone (15, 17).

Previous studies showed that 1,24(S)-(OH)2D2 is produced by Hep3B cells, a human hepatoma cell line that also has vitamin D-25-hydroxylase activity (5). A characteristic feature was that, at low concentrations of 1{alpha}OHD2, 24-hydroxylation was less than 25-hydroxylation; whereas at higher concentrations, 24-hydroxylation was greater than 25-hydroxylation. These results are similar to those in the present study with recombinant CYP3A4. It is possible, therefore, that CYP3A4 may be responsible for some of the 24- and 25-hydroxylation of 1{alpha}OHD2 in Hep3B cells. The results indicate that the enzyme has high capacity, low specificity for 24-hydroxylation of 1{alpha}OHD2 and has low capacity, high specificity for 25-hydroxylation of 1{alpha}OHD2. Further, the results also indicate that the enzyme has low capacity and high specificity for 24- and 25-hydroxylation of 1{alpha}OHD3.

24OHD2 and 1,24(S)-(OH)2D2 are present in the circulation of humans, cows, and rats given pharmacologic doses of vitamin D2 (4, 5, 6). 24OHD2 increased serum calcium in intact, but not in nephrectomized, rats. This was interpreted to mean that the metabolite was converted to 1,24-(OH)2D2 by renal 1{alpha}-hydroxylase (4). In a human subject, serum 1,24-(OH)2D2 increased in response to treatment with PTH(1–34), an activator of the enzyme (6). Based on these findings, 24-hydroxylation of vitamin D2 and its subsequent conversion to 1,24-(OH)2D2 by 1{alpha}-hydroxylation in the kidney is thought to represent a minor pathway for metabolism of the vitamin (4, 6).

With regard to calcium metabolism, 1,24(S)-(OH)2D2 has less biologic activity than 1,25-(OH)2D3. In dose-response studies, it was less effective than 1,25-(OH)2D3 in increasing serum calcium in vitamin D-deficient rats (36) and less effective than 1,25-(OH)2D3 in increasing urinary calcium in normal rats (37). In postmenopausal women, 1{alpha}OHD2 produced only modest increases in serum calcium, even when given in large doses (38). In patients with renal failure, 1{alpha}OHD2 suppresses elevated serum immunoreactive PTH and is used for treatment of secondary hyperparathyroidism in this disorder (39).

Previous studies of site-directed mutagenesis of CYP3A4 indicate that residues 119, 301, and 373 are important for testosterone hydroxylase activity; and residues 119, 304, 305, 309, 370, and 373 are important residues for progesterone hydroxylase activity (13). In the present work with 1{alpha}OHD2 and 1{alpha}OHD3 as substrates, residues 119, 120, 301, 305, and 479 were found to be important sites for modulating 24- and/or 25-hydroxylase activity. Thus, a number of the same residues are important for hydroxylation of these compounds, as they are for testosterone and progesterone. In addition, one naturally occurring CYP3A4 variant, M445T, showed a 3-fold higher 25-hydroxylation of 1{alpha}OHD3 than wild-type. Otherwise, the variants had no effect on vitamin D metabolism.

24-Hydroxylase and 25-hydroxylase activities of recombinant CYP3A4 coexpressed in baculovirus-infected insect cells with cytochrome P450 reductase and cytochrome b5 were consistently higher than those of reconstituted recombinant CYP3A4 expressed in Escherichia coli, and the difference in activity varied with substrate. Differences in enzyme activity with different substrates also was noted earlier in studies with similar preparations of recombinant CYP3A4 and four substrates: testosterone, amitriptyline, benzphetamine, and nifedipine (40). Further, the difference was attributed to higher amounts of NADPH-450 reductase in the baculovirus-infected insect cells compared with the reconstituted CYP3A4. In keeping with these findings, almost 3-times as much reductase and 4-times as much cytochrome b5 in the recombinant CYP3A4 expressed in insect cells, compared with the reconstituted CYP3A4 expressed in Escherichia coli, were used in the present studies.

Finally, CYP2R1, a microsomal enzyme that 25-hydroxylates vitamin D2 and vitamin D3 equally, was recently discovered to be a vitamin D-25-hydroxylase (41). Its confirmation as a key vitamin D-25-hydroxylase was demonstrated by the finding of an inactivating homozygous L99P mutation of the CYP2R1 gene in a young black boy from Nigeria with isolated 25OHD deficiency and rickets (42, 43). We reported previously that CYP3A4 25-hydroxylates vitamin D2 and not vitamin D3 (9). That the patient responded to modest pharmacologic doses of vitamin D2 by increasing serum 25OHD and serum calcium and healing the bone disease is attributed to 25-hydroxylation of the vitamin by CYP3A4.

In conclusion, our studies indicate that, in addition to being a vitamin D-25-hydroxylase (9), CYP3A4 is a 24-hydroxylase for vitamin D2, 1{alpha}OHD2, and 1{alpha}OHD3 and may account for some of the 24-hydroxylase activity in human liver microsomes and circulating products of 24-hydroxylation of vitamin D2 found clinically (6). Studies of site-directed mutagenesis of the enzyme showed that a number of residues were significant sites for influencing activity of the enzyme.


   Footnotes
 
First Published Online November 16, 2004

Abbreviations: CHAPS, 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate; GC, gas chromatography; LC, liquid chromatography; MS, mass spectrometry; NADPH, reduced nicotinamide adenine dinucleotide phosphate; OHD, hydroxyvitamin D; (OH)2D, dihydroxyvitamin D; TMS, tristrimethylsilyl.

This work was supported by Grants DK56603-01A1 and GM 54995 from the National Institutes of Health.

Received May 20, 2004.

Accepted November 4, 2004.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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