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Originally published In Press as doi:10.1074/jbc.M505244200 on June 22, 2005

J. Biol. Chem., Vol. 280, Issue 34, 30511-30516, August 26, 2005
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Identification of the Amino Acid Residue of CYP27B1 Responsible for Binding of 25-Hydroxyvitamin D3 Whose Mutation Causes Vitamin D-dependent Rickets Type 1*

Keiko Yamamoto{ddagger}, Eriko Uchida§, Naoko Urushino§, Toshiyuki Sakaki¶||, Norio Kagawa**, Natsumi Sawada{ddagger}{ddagger}, Masaki Kamakura¶, Shigeaki Kato§§, Kuniyo Inouye§, and Sachiko Yamada{ddagger}¶¶

From the {ddagger}Institute of Biomaterials and Bioengineering & School of Biomedical Sciences, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan, the §Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan, the Biotechnology Research Center, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Kosugi, Toyama 939-0398, Japan, the **Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, the {ddagger}{ddagger}Laboratory of Endocrinology and Molecular Metabolism, Graduate School of Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan, and the §§Institute of Molecular and Cellular Biosciences, Tokyo University, 1-1-1 Yayoi, Bunkyo, Tokyo 113-0032, Japan

Received for publication, May 12, 2005 , and in revised form, June 22, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We previously reported the three-dimensional structure of human CYP27B1 (25-hydroxyvitamin D3 1{alpha}-hydroxylase) constructed by homology modeling. Using the three-dimensional model we studied the docking of the substrate, 25-hydroxyvitamin D3, into the substrate binding pocket of CYP27B1. In this study, we focused on the amino acid residues whose point mutations cause vitamin D-dependent rickets type 1, especially unconserved residues among mitochondrial CYPs such as Gln65 and Thr409. Recently, we successfully overexpressed mouse CYP27B1 by using a GroEL/ES co-expression system. In a mutation study of mouse CYP27B1 that included spectroscopic analysis, we concluded that in a 1{alpha}-hydroxylation process, Ser408 of mouse CYP27B1 corresponding to Thr409 of human CYP27B1 forms a hydrogen bond with the 25-hydroxyl group of 25-hydroxyvitamin D3. This is the first report that shows a critical amino acid residue recognizing the 25-hydroxyl group of the vitamin D3.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The hormonally active form of vitamin D3, 1,25-(OH)2 D3,1 plays essential roles in calcium homeostasis, immunology, and cell differentiation (1). 1,25-(OH)2D3 is produced by two-step hydroxylations at the 25-position in the liver by mitochondrial CYP27A1 and then at the 1{alpha}-position in the kidney by CYP27B1. The cDNA for CYP27B1 was first cloned in 1997 (2-5), and the sequence analysis of the CYP27B1 gene confirmed that defects in CYP27B1 cause vitamin D-dependent rickets type 1 (VDDR1). To date, 16 one-point mutants and several frameshift mutants have been reported (6-9). The mutated amino acid residues seemed to play important roles in the function of 1{alpha}-hydroxylase, such as substrate binding, activation of molecular oxygen, interaction with adrenodoxin, and folding of the P450 structure (10, 11).

To investigate the mutations in depth, spectral analyses including reduced CO-difference spectra and substrate-induced difference spectra are indispensable. However, the expression levels of wild type and CYP27B1 mutants were too low to carry out spectral analyses (11, 12). Thus, enhancement of the expression level of CYP27B1 is essential for structure-function analysis of CYP27B1. Recently, we successfully overexpressed mouse CYP27B1 by using a GroEL/ES co-expression system (13). The expression level of CYP27B1 is sufficient for the preparation of large amounts of wild type and CYP27B1 mutants for structural analyses. In addition, we successfully constructed a three-dimensional structure of CYP27B1 by the homology modeling technique using the structure of rabbit microsomal CYP2C5 as a template, which is the first solved x-ray structure as a eukaryotic CYP (14, 15). The three-dimensional model of CYP27B1 provided much information about the roles of amino acid residues at the mutated positions seen in VDDR1 patients. In this study, we focused on the mutants from VDDR1 whose mutated amino acids are not conserved among six mitochondorial P450s (Fig. 1). We demonstrate which amino acid residue is responsible for substrate binding by mutation studies that include spectral analysis of CYP27B1.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—DNA modifying enzymes, restriction enzymes, and the DNA sequencing kit were purchased from Takara Shuzo Co., Ltd. (Kyoto, Japan). Primer DNAs for mutation were purchased from GENSET KK (Kyoto, Japan) (Table I). Escherichia coli DH5{alpha} (Takara Shuzo Co.) was used as a host strain. The pKSDdl was constructed from pkk223-3 as described previously (13). The GroEL/ES expression plasmid, pGro12, was kindly given by the HSP research laboratory (Kyoto, Japan). CHAPS was purchased from Dojindo (Kumamoto, Japan). NADPH was purchased from Oriental Yeast Co. (Tokyo, Japan). Bovine adrenodoxin and NADPH-adrenodoxin reductase were kindly given by Dr. Y. Nonaka of Koshien University. 25-(OH)D3 was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Other chemicals used were of the highest quality commercially available.


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  		TABLE I
Oligonucleotides used as PCR primers to generate CYP27B1 mutants

 
Molecular Modeling and Substrate Docking—Molecular modeling and graphical manipulations were performed using SYBYL 6.9 (Tripos, St. Louis, MO). 25-Hydroxyvitamin D3 was docked into the substrate binding pocket manually.

Construction of Expression Plasmids—The expression plasmid for mouse CYP27B1 with the His tag at the C terminus, pKCHis-m1{alpha}, was constructed as described (6). The expression plasmids for CYP27B1 mutants (S408I, S408T, S408V, S408A, Q65H, Q65E, Q65A, Q65L, Q65N) were generated by the QuikChangeTM Site-directed Mutagenesis kit from Stratagene (Amsterdam, the Netherlands) according to the instruction manual. The oligonucleotide primers for mutagenesis are shown in Table I. Corrected generation of the desired mutations was confirmed by DNA sequencing.

Cultivation of the Recombinant E. coli Cells—The E. coli DH5{alpha} harboring pGro12 was transformed with the expression plasmid for wild type CYP27B1 (pKCHis-m1{alpha}) or its mutants. Recombinant E. coli cells were grown in TB media (pH 7.0) containing 50 µg/ml ampicillin and 25 µg/ml kanamycin at 26 °C under good aeration for 24 h. The induction of transcription of CYP27B1 cDNA and the GroEL/ES gene was initiated by addition of isopropyl 1-thio-{beta}-D-galactopyranoside and arabinose at a final concentration of 1 mM and 4 mg/ml, respectively. {delta}-Aminolevulinic acid was also added at a final concentration of 1 mM.

Solubilization of Wild Type and CYP27B1 Mutants by CHAPS—The recombinant E. coli cells were suspended in 100 mM Tris-HCl buffer (pH 7.4) containing 1% CHAPS, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, and 20% glycerol, and disrupted by sonication for 15 min at 4 °C. Cell debris was removed at 1,200 x g for 10 min. Then the supernatant was ultracentrifugated at 100,000 x g for 1 h at 4 °C. The resultant supernatant was used for spectral and enzymatic analyses.

Measurement of Reduced CO-difference Spectra—Reduced CO-difference spectra were measured by a Shimadzu UV-2200 spectrophotometer (Kyoto, Japan) as described previously (16). The concentration of CYP27B1 was determined from the reduced CO-difference spectrum using a difference of extinction coefficient at 446 and 490 nm of 91 mM-1 cm-1 by Omura and Sato (17).

Measurement of Substrate-induced Difference Spectra—Substrate-induced difference spectra of wild type and CYP27B1 mutants were measured in the presence of 1.0 µM 25-(OH)D3 by a Shimadzu UV-2200 spectrophotometer (Kyoto, Japan).

Western Blot Analysis of Gln65 Mutants—Anti-CYP27B1 antiserum was prepared using a purified sample of mouse CYP27B1 as antigen (13). The purified CYP27B1 (0.1 mg) was mixed with an equal volume of Freund's complete adjuvant and injected intradermally into a young male Japan White rabbit. At 2, 4, 6, and 8 weeks after the first injection, the rabbit was boosted with additional injections of 0.1 or 0.2 mg each of the antigen mixed with Freund's incomplete adjuvant.

The rabbit was bled 1 week after the final injection, and antiserum was prepared. The solubilized fractions of wild type and CYP27B1 mutants containing 0.6 µg of protein were subjected to electrophoresis on 5-20% linear gradient polyacrylamide sodium dodecyl sulfate gels, and transferred electrophoretically from the gel to poly(vinylidene difluoride) membrane. The membrane was probed with the anti-CYP27B1 antiserum mentioned above, and then reacted with horseradish peroxidase-labeled rabbit IgG. The immobilized proteins were detected by treating the membrane with a mixture of 4-chloro-1-naphtol and hydrogen peroxide.



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  		FIG. 1.
Comparison of the amino acid sequences of human CYP27B1 with other mitochondrial cytochromes P450 at the mutated positions seen in patients with VDDR1. The amino acid residues that are identical and homologous in all the CYPs are shaded dark and light, respectively. The amino acid residues at the non-conserved position are surrounded by open boxes.

 
Measurement of Hydroxylation Activity Toward 25-(OH)D3, 1{alpha}-(OH)D3, and Vitamin D3—The 1{alpha}-hydroxylation activity toward 25-(OH)D3 was measured in a reconstituted system consisting of the solubilized CYP27B1 or each of its mutants (0.5-6.0 nM), 2.0 µM adrenodoxin, 0.2 µM NADPH-adrenodoxin reductase, 0.025-1.0 µM substrate, 100 mM Tris-HCl (pH 7.4), 1 mM EDTA, 0.2% glycerol, and 0.1% CHAPS in a final volume of 0.5 ml. The 25-hydroxylation activity toward 1{alpha}-(OH)D3 was measured in a similar manner except for the concentrations of 1{alpha}-(OH)D3 (0.05-1.0 µM) and CYP27B1 (2.5 nM) or S408V (50 nM). The vitamin D3 metabolism was measured in a reconstituted system consisting of 2.0 µM adrenodoxin, 0.2 µM NADPH-adrenodoxin reductase, 46 nM of the purified sample of the wild type (13), or 50 nM of the purified sample of S408V, 1.0 µM vitamin D3, 100 mM Tris-HCl (pH 7.4), 1 mM EDTA, 0.2% glycerol, and 0.1% CHAPS. The concentration of CHAPS was determined on the basis of our previous study (13). After incubation at 37 °C for 3 min, the reaction was initiated by adding NADPH at a final concentration of 1 mM. The reaction was terminated by adding 2 ml of chloroform/methanol (3:1, v/v). After extraction, the organic phase was recovered and dried. The resulting residue was solubilized with acetonitrile and applied to HPLC under the following conditions: column, YMC-Pack ODS-AM (4.6 x 300 mm) (YMC Co., Tokyo, Japan); column temperature, 40 °C; mobile phase, linear gradient of 70-100% acetonitrile aqueous solution per 15 min; flow rate, 1.0 ml/min; UV detection, 265 nm. The kinetic parameters, Km and kcat, were calculated by the nonlinear regression analysis using the KaleidaGraph (Synergy software).

Other Methods—The concentrations of vitamin D3 derivatives were estimated by their molar extinction coefficient of 1.80 x 104 M-1 cm-1 at 264 nm (18). Total protein concentrations were determined by the Bradford method using bovine serum albumin as a standard. Mouse CYP27B1, human CYP27B1, and rabbit CYP2C5 were aligned by using ClustalW interfaced with Clustal X (version 1.81) for Windows.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Docking of 25-(OH)D3 into CYP27B1—The three-dimensional structure of CYP27B1, which was constructed and reported in our previous paper (14), gave abundant insights in regards to the function of each residue. We are greatly interested in how the CYP27B1 recognizes 25-(OH)D3 and binds it as a substrate. We noted amino acid residues whose point mutant cause VDDR1 (Fig. 1). Fig. 1 shows comparison of the residues of CYP27B1 with other human mitochondrial CYPs at the mutated positions seen in patients with VDDR1. Residues conserved in these CYPs are thought to be responsible for common roles among mitochondrial CYPs, whereas residues not conserved are thought to be involved in specific roles of each enzyme such as substrate binding. Thus, we focused on mutants Q65H, P143L, E189L, S323Y, T409I, and R429P. The three-dimensional structure of CYP27B1 demonstrated that P143L, E189L, S323Y, and R429P are responsible for protein folding as previously reported (14) (Fig. 2a). On the other hand, Gln65 and Thr409 are lining the end of broad cavity above heme in the three-dimensional model of CYP27B1 (Fig. 2a).

In the docking study of 25-(OH)D3 into the pocket, it is important to determine the substrate binding site and the conformation of 25-(OH)D3. Considering the importance of Gln65 and Thr409 whose mutations cause VDDR1, we selected the substrate binding site where 25-(OH)D3 can form the hydrogen bond between the 25-OH group and Gln65 and/or Thr409. We docked 25-(OH)D3 as follows: 1) the A-ring of 25-(OH)D3 was superimposed on 1R-camphor accommodated in the substrate binding pocket of the P450cam protein (Protein Data Bank code 1DZ4 [PDB] ) (19), because, structurally and biologically, the best characterized P450 is P450cam and we analyzed the docking modes of camphor into P450cam and found the modes being approximately conserved. In addition, P450cam belongs to the class 1 enzyme in the P450 superfamily as well as mitochondrial CYPs. 2) The side chain of 25-(OH)D3 was positioned near Gln65 and Thr409. 3) Spatial location of 25-(OH)D3 was manually adjusted so as to minimize the van der Waals bump between the substrate vitamin and the amino acid residues lining the substrate binding pocket. The resulting structure of CYP27B1 and 25-(OH)D3 complex is shown in Fig. 2a. The distances between the 25-hydroxyl group and Gln65 and the 25-hydroxyl group and Thr409 are 2.83 and 2.82 Å, respectively. This suggests that the 25-hydroxyl group forms pincer-type hydrogen bonds with Gln65 and Thr409. The 1{alpha}-hydrogen of 25-(OH)D3, which will be subjected to hydroxylation, orients to the iron atom as a constituent of heme in the CYP (Fig. 2b).



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  		FIG. 2.
a, stereo view of the complex model of human CYP27B1 and 25-(OH)D3. Overall folding of CYP27B1 is represented by a ribbon-loop drawing. Sixteen amino acid residues where point mutation causes VDDR1 (atom type color), heme (red), and 25-(OH)D3 (yellow) are depicted as a ball and stick model. b, the 25-hydroxyl group of 25-(OH)D3 forms pincer-type hydrogen bond with Gln65 (2.83 Å) and Thr409 (2.82 Å), and the hydrogen at the 1{alpha} position (cyan), which will be subjected to hydroxylation, orients to an iron atom of the heme. The distance between C(1) and iron is 4.3 Å and that between hydrogen at the 1{alpha}-position and iron is 3.3 Å. c, conformation of 25-(OH)D3 docked into the substrate binding pocket of CYP27B1. d, Gln65 may interact with Tyr87 on the {beta}-sheet but not with the 25-hydroxyl group of the substrate.

 
Conformation of 25-(OH)D3 Accommodated in CYP27B1—As shown in Fig. 2c, 25-(OH)D3 is docked with a stable conformation in which the A-ring adopts the chair form and the side chain adopts the gauche(+) conformation (43°) at C(16-17-20-22) and the following anti-conformation from C(20) to the terminal methyl group. A-ring adopts {beta}-form. In this conformation, the distance between C(1) and iron is 4.3 Å and between hydrogen at the 1{alpha}-position and iron is 3.3 Å (Fig. 2b), which are consistent with the distances observed in the crystal structures: C(5)-iron (4.2 Å) and H(5)-iron (3.2 Å) in the camphor-P450cam complex (1DZ4) (19); C(5)-iron (3.9 Å) and H(5)-iron (3.3 Å) in the camphor-P450cam complex (1AKD) (20); C(1)-iron (3.8 Å) and H(1)-iron (2.9 Å) in the androstenedione-P450eryF complex (1EUP) (21). If the A-ring of 25-(OH)D3 adopts the {alpha}-form, distances of C(1)-iron and C(2)-iron are 3.9 and 3.8 Å, respectively, resulting in the absence of a rational explanation for the selective hydroxylation that occurred at the 1{alpha}-position but not the 2-position. Thus, our docking model well explains the stereospecific hydroxylation at the 1{alpha}-position of 25-(OH)D3.

Expression of Wild Type and CYP27B1 Mutants with GroEL/ES Co-expression System—Molecular modeling study of CYP27B1 strongly suggests that Ser408 of mouse CYP27B1 corresponding to Thr409 of human CYP27B1, and/or Gln65 are involved in substrate binding (Fig. 3). To reveal each function of the mutated amino acid residues, we generated multiple forms of CYP27B1 mutants at positions 408 (409 in human CYP27B1) and 65. As shown in Fig. 4, mutant S408I showed the reduced CO-difference spectra similar to wild type. S408T and S408A, and S408V, also showed similar spectra (data not shown). The expression level of the wild type of CYP27B1 was 200-300 nmol/liter of culture, as described previously (13). The expression levels of Ser408 mutants, S408I and S408A, were nearly the same as the wild type, whereas those of S408T and S408V were higher (400-450 nmol/liter of culture). On the other hand, the expression levels of Gln65 mutants (Q65H, Q65E, Q65A, Q65L, Q65N) were too low to be determined by the reduced CO-difference spectra. However, Q65E showed a substrate-induced difference spectrum. Thus, the expression level of the Q65E hemoprotein was estimated to be 10 nmol/liter based on the assumption that Q65E shows a substrate-induced difference spectrum similar to wild type. In contrast, Western blot analysis showed that the distinct bands reacted with anti-CYP27B1 antiserum in the Gln65 mutants. The expression levels of the Gln65 mutants were not so different from that of the wild type (Fig. 5). These results suggest that most Gln65 mutants are expressed as apoproteins without a heme molecule in E. coli cells.



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  		FIG. 3.
Sequence alignment of mouse and human CYP27B1. The A to L helices are labeled as defined by Williams et al. (15). Blue boxes and green bars represent the substrate recognition site (SRS) and {alpha}-helix, respectively. Black boxes show the amino acid residues where the point mutation causes VDDR1.

 



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  		FIG. 4.
Reduced CO-difference spectra of wild type mouse CYP27B1 (—), and the mutant S408I (- - -).

 
Analysis of Substrate Binding of Wild Type and Ser408 Mutants of CYP27B1 with 25-(OH)D3—As shown in Fig. 6, substrate-induced difference spectra of wild type and CYP27B1 mutants with 25-(OH)D3 showed Type I spectra, indicating the change of spin state of heme iron of CYP27B1 from low spin to high spin. The magnitude of {Delta}A390-420 in S408T was slightly larger than the wild type of CYP27B1. In contrast, the magnitude of {Delta}A390-420 in S408V and S408A was quite small, but S408I showed no detectable spectral change. These results suggest that the substrate, 25-(OH)D3, can remove the H2O molecule as the sixth axial ligand of the heme iron of wild type CYP27B1 and mutant S408T. In addition, the hydroxyl group at the side chain of the amino acid at position 408 appears to be essential for removal of the H2O molecule. It should be noted that S408I corresponding to T409I from patients with VDDR1 cannot remove the H2O molecule by 25-(OH)D3.



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  		FIG. 5.
Western blot analysis of wild type and Gln65 mutants of mouse CYP27B1. The solubilized fractions expressing each of the mutants Q65A (lane 1), Q65E (lane 2), Q65H (lane 3), Q65L (lane 4), Q65N (lane 5), control (lane 6: prepared from the control DH5{alpha}/pGro12 cells), and the wild type (lane 7) were analyzed as described under "Experimental Procedures."

 



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  		FIG. 6.
Substrate-induced difference spectra of wild type and CYP27B1 mutants with 1.0 µM 25-(OH)D3. The difference spectra of wild type and CYP27B1 mutants were measured in 100 mM Tris-HCl buffer (pH 7.4) containing 0.1% CHAPS. Enzyme concentrations were 160 nM (wild type, S408T, S408I) and 190 nM (S408A, S408V), respectively.

 
Analysis of 1{alpha}-Hydroxylation Activity of Wild Type and CYP27B1 Mutants toward 25-(OH)D3—The 1{alpha}-hydroxylation activity toward 25-(OH)D3 was examined using solubilized CYP27B1 by CHAPS as described under "Experimental Procedures." As shown in Table II, kinetic parameters, Km and kcat, of the wild type CYP27B1 were estimated to be 0.28 µM and 23.1 min-1, respectively. The kcat and Km values of S408T were significantly lower than those of the wild type CYP27B1. However, S408T appeared to have enough activity as a 1{alpha}-hydroxylase for 25-(OH)D3 based on its kcat/Km value. On the other hand, S408A and S408V showed much smaller kcat values than the wild type. As expected by spectral analysis, S408I showed the smallest activity among the mutants. Note that S408A, S408V, and S408I showed Km values not so different from the wild type.


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  		TABLE II
Kinetic parameters of wild type and CYP27B1 mutants for 25(OH)D3 1{alpha}-hydroxylation activity

 
In the same way, 1{alpha}-hydroxylation activity toward 25-(OH)D3 of Gln65 mutants was measured. Mutant Q65E showed only a small activity, although other Gln65 mutants showed no detectable activity. The Km value of Q65E was estimated to be 0.80 µM, which is considerably higher than those of Ser408 mutants (Table II). Because the concentration of Q65E hemoprotein was not determined from reduced CO-difference spectrum probably because of its unstability, the kcat value was not determined. On the assumption that Q65E shows a substrate-induced difference spectrum similar to wild type, kcat was estimated to be 0.55 min-1.



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  		FIG. 7.
HPLC profiles of vitamin D and its metabolites by the wild type (A and B) and S408V (C and D3). The metabolites at 0 min (A and C) and 40 min (B and D) were analyzed by HPLC according to "Experimental Procedures."

 



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  		FIG. 8.
Time courses of vitamin D3 metabolism by wild type (A) and S408V (B). The amounts of metabolites of vitamin D3 at 5, 10, 15, and 20 min was measured according to "Experimental Procedures."

 
Analysis of 1{alpha}-(OH)D3 25-Hydroxylation Activity of the Wild Type and S408V—We consider that the mutant T409I of human CYP27B1 corresponds to S408I of mouse CYP27B1. However, it might be possible that the conversion of Thr to Ile corresponds to the conversion of Ser to Val, judging from their chemical structure. Thus, enzymatic properties of S408V were compared with those of the wild type. As shown in Table III, the kinetic parameters, Km and kcat, of the wild type for 1{alpha}-(OH)D3 25-hydroxylation was estimated to be 0.52 µM and 0.60 min-1, respectively. Thus, the kcat/Km value was only 1.3% 1{alpha}-(OH)D3 25-hydroxylation. Ser408 showed a similar Km value but a much smaller kcat value than wild type. These results are quite similar to those for 25-(OH)D3 1{alpha}-hydroxylation, suggesting that Ser408 is involved in the binding of not only 25-(OH)D3 but also 1{alpha}-(OH)D3.


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  		TABLE III
Kinetic parameters of wild type and mutant S408V for 1{alpha}(OH)D3 25-hydroxylation activity

 
Vitamin D3 Metabolism by the Wild Type and S408V—Fig. 7 shows the HPLC profiles of vitamin D3 by wild type CYP27B1 and S408V. Both 1{alpha}-(OH)D3 and 1-{alpha},25(OH)2D3 were detected in the metabolism by the wild type. However, 25-(OH)D3 was not detected as reported previously (13). On the other hand, S408V showed a clear peak of 25-(OH)D3 in addition to those of 1{alpha}-(OH)D3 and 1{alpha},25-(OH)2D3. LC-MS analysis confirmed that this metabolite is 25-(OH)D3 (data not shown). It is possible to assume that 25-(OH)D3 is not detected as an intermediate because of its rapid conversion to 1{alpha},25-(OH)2D3 by the wild type CYP27B1, but 25-(OH)D3 is detected because of its slow conversion by S408V. Fig. 8 shows the time courses of vitamin D3 metabolism. In the metabolism of wild type CYP27B1, 1{alpha}-(OH)D3 increased up to 10 min and thereafter reached plateau, whereas 1{alpha},25-(OH)2D3 continued increasing. On the other hand, 25-(OH)D3 was not detected as described previously (13). In contrast, 25-(OH)D3 was detected as a metabolite of vitamin D3 by S408V. As shown in Fig. 7, 25-(OH)D3 increased up to 10 min and thereafter reached plateau, whereas 1{alpha}-(OH)D3 continued increasing. On the other hand, 1{alpha},25-(OH)2D3 appeared at 10 min, and then the rate of 1{alpha},25-(OH)2D3 formation increased with increasing time. Vitamin D3 metabolism together with 25-(OH)D3 1{alpha}-hydroxylation and 1{alpha}-(OH)D3 25-hydroxylation by S408V strongly indicated that S408V has a dual pathway to produce 1{alpha},25-(OH)2D3 from vitamin D3 as shown in Fig. 9. Although 25-(OH)D3 was not detected in the wild type-dependent metabolism of vitamin D3, it is possible that the wild type has a dual pathway as well as S408V.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Kitanaka et al. (6, 7) cloned eight types of missense mutations and one nonsense mutation from Japanese VDDR1 patients, and other groups identified nine missense mutations from patients (8, 9). None of the CYP27B1 mutants expressed in mammalian cells (6) and E. coli cells (11, 12) showed 1{alpha}-hydroxylase activity toward 25-(OH)D3. Thus, the mutated amino acid residues seemed to play important roles in the function of 1{alpha}-hydroxylase. Our previous study (11) suggested that Arg107, Gly125, and Pro497 destroyed the tertiary structure of the substrate-heme pocket. It was also suggested that Arg389 and Arg453 of CYP27B1 were involved in heme-propionate binding and that Asp164 stabilized the 4-helix bundle consisting of D, E, I, and J helices, possibly by forming a salt bridge. Thr321 was found to be responsible for the activation of molecular oxygen.

As shown in Fig. 1, amino acid residues at positions 65, 143, 189, 323, 409, and 429 of human CYP27B1 were not conserved among mitochondorial P450s. Of these mutations, P143L, E189G(K,L), and R429P are assumed to disrupt protein folding because Pro and Gly residues are known to be helix breakers. In addition, S323Y in the I helix appears to play an important role in protein folding because the side chain of the amino acid residue at position 323 is oriented to the opposite side of a heme molecule, buried inside the protein structure (14). The three-dimensional structure model of CYP27B1 implied that Gln65 and/or Thr409 interacts with 25-(OH)D3, probably by forming a hydrogen bond with the 25-hydroxyl group of the substrate. We have not successfully overexpressed human CYP27B1 yet, but we have successfully overexpressed mouse CYP27B1 by using a GroEL/ES co-expression system. Thus, we generated mouse CYP27B1 mutants for Gln65 and Ser408, corresponding to Thr409 of human CYP27B1. The substitution of Ser408 to Thr did not cause significant alterations in substrate binding and 1{alpha}-hydroxylation activity toward 25-(OH)D3. However, the substitutions to Ala, Val, and Ile dramatically decreased 1{alpha}-hydroxylation activity and changed the substrate binding manner. Judging from the Km values of S408A, S408V, and S408I, these mutants have significant affinity for 25-(OH)D3. However, based on their substrate-induced manner, their binding mode of the substrate appears unsuitable for displacement of H2O as the distal ligand. Because the displacement of the H2O molecule by the substrate is essential for P450 reactions, good correlation between the magnitude of {Delta}A390-420 in Fig. 5 and kcat value in Table II is quite reasonable. Note that S408V has much lower activity than S408T. The difference in the side chain of Val from Thr is the difference between a methyl and a hydroxyl group. Thus, the hydroxyl group is responsible for substrate binding for the P450 reaction. It is possible to assume that the hydroxyl group of Ser408 of mouse CYP27B1 or Thr409 of human CYP27B1 interacts with the 25-OH group of the substrate through a hydrogen bond. The high affinity of S408A, S408V, and S408I for 25-(OH)D3 may suggest that another amino acid residue takes the place of Ser408 through a hydrogen bond with the substrate.



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  		FIG. 9.
Metabolic pathway of vitamin D3 by CYP27B1 and the mutant S408V.

 
It is noted that not only 25-(OH)D3 1{alpha}-hydroxylation but also 1{alpha}-(OH)D3 25-hydroxylation by S408V was much less than those of the wild type CYP27B1. These results suggest that the hydroxyl group of Ser408 of mouse CYP27B1 is involved in substrate binding of both substrates. Note that the substrate is fixed in the opposite direction in the substrate binding pocket of CYP27B1 between 1{alpha}- and 25-hydroxylations. Thus, it appears that the hydroxyl group of Ser408 interacts with the 1{alpha}-hydroxyl group of 1{alpha}-(OH)D3. On the other hand, 1{alpha}-hydroxylation and 25-hydroxylation activities of S408V toward vitamin D3 appear to be similar to those of the wild type, based on the time courses of the metabolites shown in Fig. 7. These results are consistent with the fact that vitamin D3 has no hydroxyl groups at positions C-1{alpha} and C-25 to interact with the hydroxyl group of the amino acid at position 408.

The significantly higher Km and lower kcat values of Q65E than the wild type are consistent with an involvement of Gln65 in binding of the substrate. However, Western blot and spectral analyses of Gln65 mutants indicated that most Gln mutants were expressed as apoproteins without heme molecules, whereas Q65E showed a small amount of hemoprotein and the activity. These results strongly suggest that Gln65 is involved in protein folding.

In this study, we revealed that Ser408 and Gln65 play important roles in substrate binding and the folding of mouse CYP27B1, respectively. The reasons why mutations T409I and Q65H of human CYP27B1 cause VDDR1 were also clearly understood. It is noteworthy that the predictions derived from the three-dimensional model showed good agreement with the experimental data. Thus, this three-dimensional model gives essential information on the structure-function relationship of CYP27B1.


   FOOTNOTES
 
* This work was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan and the Sankyo Foundation of Life Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence may be addressed: Biotechnology Research Center, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Kosugi, Toyama 939-0398, Japan. Tel.: 81-766-56-7500; Fax: 81-766-56-7500; E-mail: tsakaki{at}pu-toyama.ac.jp.

¶¶ To whom correspondence may be addressed: Institute of Biomaterials and Bioengineering & School of Biomedical Sciences, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan. Tel.: 81-3-5280-8036; Fax: 81-3-5280-8005; E-mail: yamada.mr{at}tmd.ac.j.

1 The abbreviations used are: 25-(OH)D3, 25-hydroxyvitamin D3; 1{alpha},25-(OH)2D3, 1{alpha},25-dihydroxyvitamin D3; VDDR1, vitamin D-dependent rickets type 1; CYP, cytochrome P450; HPLC, high performance liquid chromatography; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. Back



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TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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