Publications

• Metabolism of vitamin D3 by cytochromes P450.

  Sakaki T., Kagawa N., Yamamoto K., Inouye K.

  The vitamin D3 25-hydroxylase (CYP27A1), 25-hydroxyvitamin D3 1alpha-hydroxylase (CYP27B1) and 1alpha,25-dihydroxyvitamin D3 24-hydroxylase (CYP24A1) are members of the cytochrome P450 superfamily, and key enzymes of vitamin D3 metabolism. Using the heterologous expression in E. coli, enzymatic pr ... >> More

  The vitamin D3 25-hydroxylase (CYP27A1), 25-hydroxyvitamin D3 1alpha-hydroxylase (CYP27B1) and 1alpha,25-dihydroxyvitamin D3 24-hydroxylase (CYP24A1) are members of the cytochrome P450 superfamily, and key enzymes of vitamin D3 metabolism. Using the heterologous expression in E. coli, enzymatic properties of the P450s were recently investigated in detail. Upon analyses of the metabolites of vitamin D3 by the reconstituted system, CYP27A1 surprisingly produced at least seven forms of minor metabolites including 1alpha,25(OH)2D3 in addition to the major metabolite 25(OH)D3. These results indicated that human CYP27A1 catalyzes multiple reactions involved in the vitamin D3 metabolism. In contrast, CYP27B1 only catalyzes the hydroxylation at C-1alpha position of 25(OH)D3 and 24R,25(OH)2D3. Enzymatic studies on substrate specificity of CYP27B1 suggest that the 1alpha-hydroxylase activity of CYP27B1 requires the presence of 25-hydroxyl group of vitamin D3 and is enhanced by 24-hydroxyl group while the presence of 23-hydroxyl group greatly reduced the activity. Eight types of missense mutations in the CYP27B1 gene found in vitamin D-dependent rickets type I (VDDR-I) patients completely abolished the 1alpha-hydroxylase activity. A three-dimensional model of CYP27B1 structure simulated on the basis of the crystal structure of rabbit CYP2C5 supports the experimental data from mutagenesis study of CYP27B1 that the mutated amino acid residues may be involved in protein folding, heme-propionate binding or activation of molecular oxygen. CYP24A1 expressed in E. coli showed a remarkable metabolic processes of 25(OH)D3 and 1alpha,25(OH)2D3. Rat CYP24A1 catalyzed six sequential monooxygenation reactions that convert 1alpha,25(OH)2D3 into calcitroic acid, a known final metabolite of C-24 oxidation pathway. In addition to the C-24 oxidation pathway, human CYP24A1 catalyzed also C-23 oxidation pathway to produce 1alpha,25(OH)2D3-26,23-lactone. Surprisingly, more than 70 % of the vitamin D metabolites observed in a living body were found to be the products formed by the activities of CYP27A1, CYP27B1 and CYP24A1. The species-based difference was also observed in the metabolism of vitamin D analogs by CYP24A1, suggesting that the recombinant system for human CYP24A1 may be of great use for the prediction of the metabolism of vitamin D analogs in humans. << Less

  Front. Biosci. 10:119-134(2005) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Single A326G mutation converts human CYP24A1 from 25-OH-D3-24-hydroxylase into -23-hydroxylase, generating 1alpha,25-(OH)2D3-26,23-lactone.

  Prosser D.E., Kaufmann M., O'Leary B., Byford V., Jones G.

  Studies of 25-hydroxyvitamin D(3)-24-hydroxylase (CYP24A1) have demonstrated that it is a bifunctional enzyme capable of the 24-hydroxylation of 1alpha,25-(OH)(2)D(3), leading to the excretory form, calcitroic acid, and 23-hydroxylation, culminating in 1alpha,25-(OH)(2)D(3)-26,23-lactone. The degr ... >> More

  Studies of 25-hydroxyvitamin D(3)-24-hydroxylase (CYP24A1) have demonstrated that it is a bifunctional enzyme capable of the 24-hydroxylation of 1alpha,25-(OH)(2)D(3), leading to the excretory form, calcitroic acid, and 23-hydroxylation, culminating in 1alpha,25-(OH)(2)D(3)-26,23-lactone. The degree to which CYP24A1 performs either 23- or 24-hydroxylation is species-dependent. In this paper, we show that the human enzyme that predominantly 24-hydroxylates its substrate differs from the opossum enzyme that 23-hydroxylates it at only a limited number of amino acid residues. Mutagenesis of the human form at a single substrate-binding residue (A326G) dramatically changes the regioselectivity of the enzyme from a 24-hydroxylase to a 23-hydroxylase, whereas other modifications have no effect. Ala-326 is located in the I-helix, close to the terminus of the docked 25-hydroxylated side chain in a CYP24A1 homology model, a result that we interpret indicates that substitution of a glycine at 326 provides extra space for the side chain of the substrate to move deeper into the pocket and place it in a optimal stereochemical position for 23-hydroxylation. We discuss the physiological ramifications of these results for species possessing the A326G substitution, as well as implications for optimal vitamin D analog design. << Less

  Proc Natl Acad Sci U S A 104:12673-12678(2007) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Metabolism of A-ring diastereomers of 1alpha,25-dihydroxyvitamin D3 by CYP24A1.

  Kusudo T., Sakaki T., Abe D., Fujishima T., Kittaka A., Takayama H., Hatakeyama S., Ohta M., Inouye K.

  The metabolism of 1alpha,25(OH)(2)D(3) (1alpha,3beta) and its A-ring diastereomers, 1beta,25(OH)(2)D(3) (1beta,3beta), 1alpha,25(OH)(2)-3-epi-D(3) (1alpha,3alpha), and 1beta,25(OH)(2)-3-epi-D(3) (1beta,3alpha), was examined to compare the substrate specificity and reaction specificity of CYP24A1 b ... >> More

  The metabolism of 1alpha,25(OH)(2)D(3) (1alpha,3beta) and its A-ring diastereomers, 1beta,25(OH)(2)D(3) (1beta,3beta), 1alpha,25(OH)(2)-3-epi-D(3) (1alpha,3alpha), and 1beta,25(OH)(2)-3-epi-D(3) (1beta,3alpha), was examined to compare the substrate specificity and reaction specificity of CYP24A1 between humans and rats. The ratio between C-23 and C-24 oxidation pathways in human CYP24A1-dependent metabolism of (1alpha,3alpha) and (1beta,3alpha) was 1:1, although the ratio for (1alpha,3beta) and (1beta,3beta) was 1:4. These results indicate that the orientation of the hydroxyl group at the C-3 position determines the ratio between C-23 and C-24 oxidation pathways. A remarkable increase of metabolites in the C-23 oxidation pathway was also observed in rat CYP24A1-dependent metabolism. The binding affinity of human CYP24A1 for A-ring diastereomers was (1alpha,3beta)>(1alpha,3alpha)>(1beta,3beta)>(1beta,3alpha), indicating that both hydroxyl groups at C-1 and C-3 positions significantly affect substrate-binding. The information obtained in this study is quite useful for understanding substrate recognition of CYP24A1 and designing new vitamin D analogs. << Less

  Biochem Biophys Res Commun 321:774-782(2004) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Structure-function analysis of vitamin D 24-hydroxylase (CYP24A1) by site-directed mutagenesis: amino acid residues responsible for species-based difference of CYP24A1 between humans and rats.

  Hamamoto H., Kusudo T., Urushino N., Masuno H., Yamamoto K., Yamada S., Kamakura M., Ohta M., Inouye K., Sakaki T.

  Our previous studies revealed the species-based difference of CYP24A1-dependent vitamin D metabolism. Although human CYP24A1 catalyzes both C-23 and C-24 oxidation pathways, rat CYP24A1 shows almost no C-23 oxidation pathway. We tried to identify amino acid residues that cause the species-based di ... >> More

  Our previous studies revealed the species-based difference of CYP24A1-dependent vitamin D metabolism. Although human CYP24A1 catalyzes both C-23 and C-24 oxidation pathways, rat CYP24A1 shows almost no C-23 oxidation pathway. We tried to identify amino acid residues that cause the species-based difference by site-directed mutagenesis. In the putative substrate-binding regions, amino acid residue of rat CYP24A1 was converted to the corresponding residue of human CYP24A1. Among eight mutants examined, T416M and I500T showed C-23 oxidation pathway. In addition, the mutant I500F showed quite a different metabolism of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] from both human and rat CYP24A1. These results strongly suggest that the amino acid residues at positions 416 and 500 play a crucial role in substrate binding and greatly affect substrate orientation. A three-dimensional model of CYP24A1 indicated that the A-ring and triene part of 1alpha,25(OH)2D3 could be located close to amino acid residues at positions 416 and 500, respectively. Our findings provide useful information for the development of new vitamin D analogs for clinical use. << Less

  Mol. Pharmacol. 70:120-128(2006) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Dual metabolic pathway of 25-hydroxyvitamin D3 catalyzed by human CYP24.

  Sakaki T., Sawada N., Komai K., Shiozawa S., Yamada S., Yamamoto K., Ohyama Y., Inouye K.

  Human 25-hydroxyvitamin D3 (25(OH)D3) 24-hydroxylase (CYP24) cDNA was expressed in Escherichia coli, and its enzymatic and spectral properties were revealed. The reconstituted system containing the membrane fraction prepared from recombinant E. coli cells, adrenodoxin and adrenodoxin reductase was ... >> More

  Human 25-hydroxyvitamin D3 (25(OH)D3) 24-hydroxylase (CYP24) cDNA was expressed in Escherichia coli, and its enzymatic and spectral properties were revealed. The reconstituted system containing the membrane fraction prepared from recombinant E. coli cells, adrenodoxin and adrenodoxin reductase was examined for the metabolism of 25(OH)D3, 1alpha,25(OH)2D3 and their related compounds. Human CYP24 demonstrated a remarkable metabolism consisting of both C-23 and C-24 hydroxylation pathways towards both 25(OH)D3 and 1alpha,25(OH)2D3, whereas rat CYP24 showed almost no C-23 hydroxylation pathway [Sakaki, T. Sawada, N. Nonaka, Y. Ohyama, Y. & Inouye, K. (1999) Eur. J. Biochem. 262, 43-48]. HPLC analysis and mass spectrometric analysis revealed that human CYP24 catalyzed all the steps of the C-23 hydroxylation pathway from 25(OH)D3 via 23S, 25(OH)2D3, 23S,25,26(OH)3D3 and 25(OH)D3-26,23-lactol to 25(OH)D3-26, 23-lactone in addition to the C-24 hydroxylation pathway from 25(OH)D3 via 24R,25(OH)2D3, 24-oxo-25(OH)D3, 24-oxo-23S,25(OH)2D3 to 24,25,26,27-tetranor-23(OH)D3. On 1alpha,25(OH)2D3 metabolism, similar results were observed. These results strongly suggest that the single enzyme human CYP24 is greatly responsible for the metabolism of both 25(OH)D3 and 1alpha,25(OH)2D3. We also succeeded in the coexpression of CYP24, adrenodoxin and NADPH-adrenodoxin reductase in E. coli. Addition of 25(OH)D3 to the recombinant E. coli cell culture yielded most of the metabolites in both the C-23 and C-24 hydroxylation pathways. Thus, the E. coli expression system for human CYP24 appears quite useful in predicting the metabolism of vitamin D analogs used as drugs. << Less

  Eur. J. Biochem. 267:6158-6165(2000) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Evidence for the activation of 1alpha-hydroxyvitamin D2 by 25-hydroxyvitamin D-24-hydroxylase: delineation of pathways involving 1alpha,24-dihydroxyvitamin D2 and 1alpha,25-dihydroxyvitamin D2.

  Masuda S., Strugnell S.A., Knutson J.C., St-Arnaud R., Jones G.

  While current dogma argues that vitamin D prodrugs require side-chain activation by liver enzymes, recent data suggest that hydroxylation may also occur extrahepatically. We used keratinocytes and recombinant human enzyme to test if the 25-hydroxyvitamin D-24-hydroxylase (CYP24A1) is capable of ta ... >> More

  While current dogma argues that vitamin D prodrugs require side-chain activation by liver enzymes, recent data suggest that hydroxylation may also occur extrahepatically. We used keratinocytes and recombinant human enzyme to test if the 25-hydroxyvitamin D-24-hydroxylase (CYP24A1) is capable of target cell activation and inactivation of a model prodrug, 1alpha-hydroxyvitamin D2 (1alpha(OH)D2) in vitro. Mammalian cells stably transfected with CYP24A1 (V79-CYP24A1) converted 1alpha(OH)D2 to a series of metabolites similar to those observed in murine keratinocytes and the human cell line HPK1A-ras, confirming the central role of CYP24A1 in metabolism. Products of 1alpha(OH)D2 included the active metabolites 1alpha,24-dihydroxyvitamin D2 (1alpha,24(OH)2D2) and 1alpha,25-dihydroxyvitamin D2 (1alpha,25(OH)2D2); the formation of both indicating the existence of distinct activation pathways. A novel water-soluble metabolite, identified as 26-carboxy-1alpha,24(OH)2D2, was the presumed terminal degradation product of 1alpha(OH)D2 synthesized by CYP24A1 via successive 24-hydroxylation, 26-hydroxylation and further oxidation at C-26. This acid was absent in keratinocytes from Cyp24a1 null mice. Slower clearance rates of 1alpha(OH)D2 and 1alpha,24(OH)2D2 relative to 1alpha,25(OH)2D2 and 1alpha,25(OH)2D3 were noted, arguing for a role of 24-hydroxylated metabolites in the altered biological activity profile of 1alpha(OH)D2. Our findings suggest that CYP24A1 can activate and inactivate vitamin D prodrugs in skin and other target cells in vitro, offering the potential for treatment of hyperproliferative disorders such as psoriasis by topical administration of these prodrugs. << Less

  Biochim Biophys Acta 1761:221-234(2006) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Kinetic analysis of human CYP24A1 metabolism of vitamin D via the C24-oxidation pathway.

  Tieu E.W., Tang E.K., Tuckey R.C.

  CYP24A1 is the multicatalytic cytochrome P450 responsible for the catabolism of vitamin D via the C23- and C24-oxidation pathways. We successfully expressed the labile human enzyme in Escherichia coli and partially purified it in an active state that permitted detailed characterization of its meta ... >> More

  CYP24A1 is the multicatalytic cytochrome P450 responsible for the catabolism of vitamin D via the C23- and C24-oxidation pathways. We successfully expressed the labile human enzyme in Escherichia coli and partially purified it in an active state that permitted detailed characterization of its metabolism of 1,25-dihydroxyvitamin D3 [1,25(OH)2 D3] and the intermediates of the C24-oxidation pathway in a phospholipid-vesicle reconstituted system. The C24-oxidation pathway intermediates, 1,24,25-trihydroxyvitamin D3, 24-oxo-1,25-dihydroxyvitamin D3, 24-oxo-1,23,25-trihydroxyvitamin D3 and tetranor-1,23-dihydroxyvitamin D3, were enzymatically produced from 1,25(OH)2 D3 using rat CYP24A1. Both 1,25(OH)2 D3 and 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3 were found to partition strongly into the phospholipid bilayer when in aqueous medium. Changes to the phospholipid concentration did not affect the kinetic parameters for the metabolism of 1,25(OH)2 D3 by CYP24A1, indicating that it is the concentration of substrates in the membrane phase (mol substrate·mol phospholipid(-1) ) that determines their rate of metabolism. CYP24A1 exhibited Km values for the different C24-intermediates ranging from 0.34 to 15 mmol·mol phospholipid(-1) , with 24-oxo-1,23,25-trihydroxyvitamin D3 [24-oxo-1,23,25(OH)3 D3] displaying the lowest and 1,24,25-trihydroxyvitamin D3 [1,24,25(OH)3 D3] displaying the highest. The kcat values varied by up to 3.8-fold, with 1,24,25(OH)3 D3 displaying the highest kcat (34 min(-1) ) and 24-oxo-1,23,25(OH)3 D3 the lowest. The data show that the cleavage of the side chain of 24-oxo-1,23,25(OH)3 D3 occurs with the highest catalytic efficiency (kcat /Km ) and produces 1-hydroxy-23-oxo-24,25,26,27-tetranorvitamin D3 and not 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3, as the primary product. These kinetic analyses also show that intermediates of the C24-oxidation pathway effectively compete with precursor substrates for binding to the active site of the enzyme, which manifests as an accumulation of intermediates, indicating that they dissociate after each catalytic step. << Less

  FEBS J. 281:3280-3296(2014) [PubMed] [EuropePMC]

  This publication is cited by 9 other entries.

• Novel metabolism of 1 alpha,25-dihydroxyvitamin D3 with C24-C25 bond cleavage catalyzed by human CYP24A1.

  Sawada N., Kusudo T., Sakaki T., Hatakeyama S., Hanada M., Abe D., Kamao M., Okano T., Ohta M., Inouye K.

  Our previous study revealed that human CYP24A1 catalyzes a remarkable metabolism consisting of both C-23 and C-24 hydroxylation pathways that used both 25(OH)D(3) and 1alpha,25(OH)(2)D(3) as substrates, while rat CYP24A1 showed extreme predominance of the C-24 over C-23 hydroxylation pathway [Saka ... >> More

  Our previous study revealed that human CYP24A1 catalyzes a remarkable metabolism consisting of both C-23 and C-24 hydroxylation pathways that used both 25(OH)D(3) and 1alpha,25(OH)(2)D(3) as substrates, while rat CYP24A1 showed extreme predominance of the C-24 over C-23 hydroxylation pathway [Sakaki, T., Sawada, N., Komai, K., Shiozawa, S., Yamada, S., Yamamoto, K., Ohyama, Y. and Inouye, K. (2000) Eur. J. Biochem. 267, 6158-6165]. In this study, by using the Escherichia coli expression system for human CYP24A1, we identified 25,26,27-trinor-23-ene-D(3) and 25,26,27-trinor-23-ene-1alpha(OH)D(3) as novel metabolites of 25(OH)D(3) and 1alpha,25(OH)(2)D(3), respectively. These metabolites appear to be closely related to the C-23 hydroxylation pathway, because human CYP24A1 produces much more of these metabolites than does rat CYP24A1. We propose that the C(24)-C(25) bond cleavage occurs by a unique reaction mechanism including radical rearrangement. Namely, after hydrogen abstraction of the C-23 position of 1alpha,25(OH)(2)D(3), part of the substrate-radical intermediate is converted into 25,26,27-trinor-23-ene-1alpha(OH)D(3), while a major part of them is converted into 1alpha,23,25(OH)(3)D(3). Because the C(24)-C(25) bond cleavage abolishes the binding affinity of 1alpha,25(OH)D(3) for the vitamin D receptor, this reaction is quite effective for inactivation of 1alpha,25(OH)D(3). << Less

  Biochemistry 43:4530-4537(2004) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Enzymes involved in the activation and inactivation of vitamin D.

  Prosser D.E., Jones G.

  Six cytochrome P450 (CYP) isoforms have been shown to hydroxylate vitamin D. Four of these, CYP27A1, CYP2R1, CYP3A4 and CYP2J3, are candidates for the enzyme vitamin D 25-hydroxylase that is involved in the first step of activation. The highly regulated, renal enzyme 25-hydroxyvitamin D-1alpha-hyd ... >> More

  Six cytochrome P450 (CYP) isoforms have been shown to hydroxylate vitamin D. Four of these, CYP27A1, CYP2R1, CYP3A4 and CYP2J3, are candidates for the enzyme vitamin D 25-hydroxylase that is involved in the first step of activation. The highly regulated, renal enzyme 25-hydroxyvitamin D-1alpha-hydroxylase contains the component CYP27B1, which completes the activation pathway to the hormonal form 1alpha,25-dihydroxyvitamin D(3). A five-step inactivation pathway from 1alpha,25-(OH)(2)D(3) to calcitroic acid is attributed to a single multifunctional CYP, CYP24A1, which is transcriptionally induced in vitamin D target cells by the action of 1alpha,25-(OH)(2)D(3). On the basis of alignments and crystal structures of other CYPs, homology models of vitamin-D-related CYPs have been generated. Two human forms of rickets caused by mutations of CYP2R1 and CYP27B1, as well as mouse knockout models of CYP27A1, CYP27B1 and CYP24A1, are helping us to establish the full in vivo physiological roles of the vitamin-D-related hydroxylases. << Less

  Trends Biochem Sci 29:664-673(2004) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Human 25-hydroxyvitamin D3-24-hydroxylase, a multicatalytic enzyme.

  Beckman M.J., Tadikonda P., Werner E., Prahl J., Yamada S., DeLuca H.F.

  Human 25-hydroxyvitamin D-24-hydroxylase has been expressed in Spodoptera frugiperda (Sf21) insect cells using the previously cloned cDNA in baculovirus (AcNPV-P450cc24). The activity of recombinant h-P450cc24 required adrenodoxin, adrenodoxin reductase, and NADPH. Incubation of this reconstituted ... >> More

  Human 25-hydroxyvitamin D-24-hydroxylase has been expressed in Spodoptera frugiperda (Sf21) insect cells using the previously cloned cDNA in baculovirus (AcNPV-P450cc24). The activity of recombinant h-P450cc24 required adrenodoxin, adrenodoxin reductase, and NADPH. Incubation of this reconstituted system with 25-OH-[26,27-(3)H]D3 substrate produced several metabolites that were resolved on a normal-phase cyano HPLC system. These products exactly comigrated with authentic standards for 24-oxo-25-OH-D3, 23(S),25-(OH)2D3, 24(R),25-(OH)2D3, and 24-oxo-23(S),25-(OH)2D3. The soluble proteins from Sf21 cells infected with wild-type baculovirus produced neither 24,25-(OH)2D3 nor any of the other 25-OH-D3 metabolites. The products were isolated and subjected to a normal-phase amino HPLC for further separation, purification, and characterization. Comigration on two HPLC systems, periodate cleavage reactions, and NaBH4 reduction established clearly the identity of these metabolites. Incubation of recombinant h-P450cc24 with 25-OH-[3 alpha-3H]D3 led to the isolation of an additional product that comigrated with 24,25,26,27-tetranor-23-OH-D3. Treatment of putative 24,25,26,27-tetranor-23-OH-[3 alpha-3H]D3 with acetic anhydride changed its migration on amino HPLC to a less polar position, indicating acetylation of a hydroxyl group(s). These data demonstrate conclusively that h-P450cc24 is a multicatalytic enzyme catalyzing most, if not all, of the reactions in the C-24/C-23 pathway of 25-OH-D3 metabolism. It is likely that this enzyme by itself converts 25-OH-D3 and 1,25-(OH)2D3 to one of its final excretion products. << Less

  Biochemistry 35:8465-8472(1996) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.