23(S),25-dihydroxy-24-oxovitamin D3

• 分子结构分类: 有机化合物 - 脂质和类脂质分子 - 类固醇和类固醇衍生物
• 英文名称: 23(S),25-dihydroxy-24-oxovitamin D3
• 英文别名: (4S,6R)-6-[(1R,3aS,4E,7aR)-4-[(2Z)-2-[(5S)-5-hydroxy-2-methylidenecyclohexylidene]ethylidene]-7a-methyl-2,3,3a,5,6,7-hexahydro-1H-inden-1-yl]-2,4-dihydroxy-2-methylheptan-3-one
• 化学式: C₂₇H₄₂O₄
• 分子量: 430.6
• InChiKey: LYVJVKJTSXESPC-JDHDJSQBSA-N

计算性质

• 辛醇/水分配系数(LogP)：
  4.4
• 重原子数：
  31
• 可旋转键数：
  6
• 环数：
  3.0
• sp3杂化的碳原子比例：
  0.74
• 拓扑面积：
  77.8
• 氢给体数：
  3
• 氢受体数：
  4

反应信息

• 作为产物：
  描述：

  (6R)-6-[(1R,3aS,4E,7aR)-4-[(2Z)-2-[(5R)-5-羟基-2-亚甲基环己亚基]亚乙基]-7a-甲基-2,3,3a,5,6,7-六氢-1H-茚-1-基]-2-羟基-2-甲基庚烷-3-酮 、 氢(+1)阳离子 、 氧气 、 [2Fe-2S]¹⁺ 生成 23(S),25-dihydroxy-24-oxovitamin D3 、 水 、 [2Fe-2S]²⁺
  参考文献：
  名称：

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

  摘要：

  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, 1α,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 1α,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 1α,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 1α,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.

  DOI：

  10.1046/j.1432-1327.2000.01680.x

文献信息

• Human 25-Hydroxyvitamin D₃-24-Hydroxylase, a Multicatalytic Enzyme
  作者：Matthew J. Beckman、Praveen Tadikonda、Elizabeth Werner、Jean Prahl、Sachiko Yamada、Hector F. DeLuca

  DOI：10.1021/bi960658i

  日期：1996.1.1

  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-H-3]D-3 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-D-3, 23(S),25-(OH)(2)D-3, 24(R),25-(OH)(2)D-3, and 24-oxo-23(S),25-(OH)(2)D-3. The soluble proteins from Sf21 cells infected with wild-type baculovirus produced neither 24,25-(OH)(2)D-3 nor any of the other 25-OH-D-3 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-H-3]D-3 led to the isolation of an additional product that comigrated with 24,25,26,27-tetranor-23-OH-D-3. Treatment of putative 24,25,26,27-tetranor-23-OH-[3 alpha-H-3]D-3 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-D-3 metabolism. It is likely that this enzyme by itself converts 25-OH-D-3 and 1,25-(OH)(2)D-3 to one of its final excretion products.
• Dual metabolic pathway of 25-hydroxyvitamin D3 catalyzed by human CYP24
  作者：Toshiyuki Sakaki、Natsumi Sawada、Koichiro Komai、Shunichi Shiozawa、Sachiko Yamada、Keiko Yamamoto、Yoshihiko Ohyama、Kuniyo Inouye

  DOI：10.1046/j.1432-1327.2000.01680.x

  日期：2000.10

  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, 1α,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 1α,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 1α,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 1α,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.
• Kinetic analysis of human CYP24A1 metabolism of vitamin D via the C24-oxidation pathway
  作者：Elaine W. Tieu、Edith K. Y. Tang、Robert C. Tuckey

  DOI：10.1111/febs.12862

  日期：2014.7

  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)2D3] 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)2D3 using rat CYP24A1. Both 1,25(OH)2D3 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)2D3 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)3D3] displaying the lowest and 1,24,25‐trihydroxyvitamin D3 [1,24,25(OH)3D3] displaying the highest. The kcat values varied by up to 3.8‐fold, with 1,24,25(OH)3D3 displaying the highest kcat (34 min−1) and 24‐oxo‐1,23,25(OH)3D3 the lowest. The data show that the cleavage of the side chain of 24‐oxo‐1,23,25(OH)3D3 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.