17-ethyl-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene | 26856-62-2

• 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: 17-ethyl-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene
• CAS: 26856-62-2
• 化学式: C₂₁H₃₆
• 分子量: 288.5
• InChiKey: JWMFYGXQPXQEEM-UHFFFAOYSA-N

计算性质

• 辛醇/水分配系数(LogP)：
  8.4
• 重原子数：
  21
• 可旋转键数：
  1
• 环数：
  4.0
• sp3杂化的碳原子比例：
  1.0
• 拓扑面积：
  0
• 氢给体数：
  0
• 氢受体数：
  0

反应信息

• 作为反应物：
  描述：

  17-ethyl-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene 、 氧气 、 1-Deoxy-1-(7,8-dimethyl-2,4-dioxidobenzo[g]pteridin-10(5H)-yl)-5-O-phosphonopentitol 生成 2-(10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)ethanol 、 氢(+1)阳离子 、 水 、 FMN
  参考文献：
  名称：

  肾上腺匀浆的分馏提取物合成C14-17-羟基-11-脱氧皮质酮和17-羟基皮质酮。

  摘要：

  DOI：

  10.1016/0003-9861(53)90378-6

文献信息

• Engineering, expression, and purification of “soluble” human cytochrome P45017α and its functional characterization
  作者：T. A. Pechurskaya、O. P. Lukashevich、A. A. Gilep、S. A. Usanov

  DOI：10.1134/s0006297908070092

  日期：2008.7

  To engineer a “soluble” form of membrane-bound cytochrome P45017α (CYP17)—a key enzyme in steroid hormone biosynthesis—in the present work we have built a computer model of the tertiary structure of the hemeprotein, identified the surface hydrophobic amino acid residues, substituted these residues for more hydrophilic ones, and expressed and purified hydrophilized forms of CYP17. We have constructed and purified the following mutant forms of human CYP17: CYP17dH (CYP17 with deleted hydrophobic N-terminal sequence (δ23)) and CYP17mod (CYP17dH with substituted cluster of hydrophobic amino acid residues in the region of the FG-loop). Removal of the N-terminal sequence responsible for interaction with the membrane does not dramatically change the association of the protein with the membrane. However, CYP17mod containing hydrophilic FG-loop is mostly localized in the cytosolic fraction. Thus, in the present work we for the first time engineered a “soluble” form of the usually membrane-bound human CYP17 that is not bound to membrane. The expression degree of CYP17mod is approximately 900 nmol/liter of culture. The hemeprotein can be purified to apparent homogeneity without using detergents at any purification step. It is shown that replacement of hydrophobic amino acid residues in the FG-loop region does not change the metabolic profile during hydroxylation of steroids that is characteristic for wild type CYP17. Besides, the modification of the hemeprotein does not affect the affinity of CYP17 to steroid substrates. The engineered “soluble” form of human CYP17 is used as a subject for crystallization of the hemeprotein.

  为了设计一种“可溶性”的膜结合细胞色素P45017α（CYP17）形式（一种类固醇激素生物合成中的关键酶），在本工作中，我们构建了血红素蛋白三级结构的计算机模型，确定了表面疏水性氨基酸残基，用更亲水的氨基酸残基替换这些残基，并表达和纯化了亲水化的CYP17形式。我们构建并纯化了以下人类CYP17突变形式：CYP17dH（CYP17，去除了疏水性N-末端序列（δ23））和CYP17mod（CYP17dH，在FG环区域替换了疏水性氨基酸残基簇）。去除负责与膜相互作用的N-末端序列不会显着改变蛋白质与膜的结合。然而，含有亲水性FG环的CYP17mod主要定位于细胞质部分。因此，在本工作中，我们首次设计了一种通常与膜结合的人类CYP17的“可溶性”形式，这种形式不与膜结合。CYP17mod的表达度约为900 nmol/升培养物。血红素蛋白可以在任何纯化步骤中不使用洗涤剂纯化到明显的均匀性。结果表明，在FG环区域替换疏水性氨基酸残基不会改变类固醇羟基化过程中的代谢特征，这是野生型CYP17的特征。此外，血红
• Studies of the Human Testis. XIII. Properties of Nicotinamide Adenine Dinucleotide (Reduced Form) linked 17α-Hydroxyiation*
  作者：KEN-ICHIRO YOSHIDA、HIROYUKI OSHIMA、PHILIP TROEN

  DOI：10.1210/jcem-50-5-895

  日期：1980.5

  A NADH-linked 17αhydroxylation of progesterone was examined using the washed microsomefraction prepared from human testes. The addition of NADP revealed no enhancement of 17α-hydroxylation, indicating no transhydrogenation from NADH to NADP in the microsome fraction. The results further substantiate the presence of NADH-linked activity of 17α-hydroxylase in addition to NADPH-linked activity. The Km of 17α-hydroxylase for NADH was calculated as 4.3×10-5 M at pH 7.4 and 37 C. The optimal pH of 17α-hydroxylase was 7.7 in the presence of NADH and 7.9 in the presence of NADPH. An additive increase in the amount of product 17α-hydroxyprogesterone was observed by adding NADH to the incubation medium containing an excess amount of NADPH. The data suggest that there are two distinct active sites for 17α-hydroxylation. Furthermore,the inhibition of NADH-linked 17α-hydroxylation by carbon monoxide indicates the involvement of cytochrome P450 in the electron transport system for the NADH-linked 17αhydroxylation.

  使用从人类睾丸中提取的洗涤微粒体片段，对孕酮的NADH相关17α羟化作用进行了研究。加入NADP后，17α羟化作用并未增强，表明微粒体片段中不存在从NADH到NADP的转氢作用。研究结果进一步证实，除了NADPH相关活性外，还存在NADH相关17α羟化酶活性。在pH 7.4和37℃下，NADH的17α羟化酶Km值计算为4.3×10-5 M。在NADH存在的情况下，17α羟化酶的最佳pH值为7.7；在NADPH存在的情况下，最佳pH值为7.9。在含有过量NADPH的培养基中加入NADH后，17α羟孕酮的产物量出现增加。数据表明，17α羟化作用存在两个不同的活性位点。此外，一氧化碳对NADH相关17α羟化作用的抑制表明，细胞色素P450参与了NADH相关17α羟化作用的电子传递系统。
• Human Cytochrome P450 21A2, the Major Steroid 21-Hydroxylase
  作者：Pradeep S. Pallan、Chunxue Wang、Li Lei、Francis K. Yoshimoto、Richard J. Auchus、Michael R. Waterman、F. Peter Guengerich、Martin Egli

  DOI：10.1074/jbc.m115.646307

  日期：2015.5

  Cytochrome P450 (P450) 21A2 is the major steroid 21-hydroxylase, and deficiency of this enzyme is involved in similar to 95% of cases of human congenital adrenal hyperplasia, a disorder of adrenal steroidogenesis. A structure of the bovine enzyme that we published previously (Zhao, B., Lei, L., Kagawa, N., Sundaramoorthy, M., Banerjee, S., Nagy, L. D., Guengerich, F. P., and Waterman, M. R. (2012) Three-dimensional structure of steroid 21-hydroxylase (cytochrome P450 21A2) with two substrates reveals locations of disease-associated variants. J. Biol. Chem. 287, 10613-10622), containing two molecules of the substrate 17 alpha-hydroxyprogesterone, has been used as a template for understanding genetic deficiencies. We have now obtained a crystal structure of human P450 21A2 in complex with progesterone, a substrate in adrenal 21-hydroxylation. Substrate binding and release were fast for human P450 21A2 with both substrates, and pre-steady-state kinetics showed a partial burst but only with progesterone as substrate and not 17 alpha-hydroxyprogesterone. High intermolecular non-competitive kinetic deuterium isotope effects on both k(cat) and k(cat)/K-m, from 5 to 11, were observed with both substrates, indicative of rate-limiting C-H bond cleavage and suggesting that the juxtaposition of the C21 carbon in the active site is critical for efficient oxidation. The estimated rate of binding of the substrate progesterone (k(on) 2.4 x 10(7) M-1 s(-1)) is only similar to 2-fold greater than the catalytic efficiency (k(cat)/K-m = 1.3 x 10(7) M-1 s(-1)) with this substrate, sug-gesting that the rate of substrate binding may also be partially rate-limiting. The structure of the human P450 21A2-substrate complex provides direct insight into mechanistic effects of genetic variants.
• ——
  作者：A. A. Gilep、R. W. Estabrook、S. A. Usanov

  DOI：10.1023/a:1022101703670

  日期：——

  To elucidate the nature of substrate specificity and intrinsic mechanism of hydroxylation of steroids, in the present work we carried out molecular cloning and heterologous expression of cDNA for three new forms of cytochrome P45017alpha from species of the Bovidae family (sheep, goat, and bison), which catalyze 17alpha-hydroxylation of both progesterone (P4) or pregnenolone (P5) and 17,20-lyase reaction resulting in cleavage of side chain with formation of C-19-steroids. Recombinant cytochromes P45017alpha were expressed in E. coli as derivatives, containing a six-His tag at the C-terminal sequence that simplifies purification of the cloned heme proteins using metal-affinity chromatography. Highly purified cytochromes P45017alpha were used for determination of enzyme activity and specificity in relation to progesterone, pregnenolone, 17alpha-hydroxyprogesterone, and 17alpha-hydroxypregnenolone with registration of the kinetics of reaction product formation using HPLC. It is shown that each form of cytochrome P45017alpha is characterized by a specific profile of enzyme activity and dependence of 17,20-lyase reaction on the presence of cytochrome b(5) in the reaction mixture. The analysis of the activity of the known forms of cytochrome P45017alpha in view of the data obtained in the present work allows the division of known cytochromes P45017alpha into three main group: group A (pig, hamster, rat), cytochromes P45017alpha catalyze the reaction of 17alpha-hydroxylation of both P4 and P5 steroids and the 17,20-lyase reaction of 17alpha-hydroxyprogesterone and 17alpha-hydroxypregnenolone; group B (human, bovine, sheep, goat, and bison), cytochromes P45017alpha, which have no or have insignificant 17,20-lyase activity in relation to 17alpha-hydroxyprogesterone; group C (guinea pig), cytochrome P45017alpha which either has no or has insignificant 17,20-lyase activity on transformation 17alpha-hydroxypregnenolone to dehydroepiandrosterone.
• Functional expression and characterisation of human cytochrome P45017α in Pichia pastoris
  作者：Norbert W. Kolar、Amanda C. Swart、J. Ian Mason、Pieter Swart

  DOI：10.1016/j.jbiotec.2007.02.003

  日期：2007.5

  Human cytochrome P45017 alpha (CYP17), present in mammalian adrenal and gonadal tissues, catalyses both steroid 17-hydroxylation and C17,20 lyase reactions, producing intermediates for the glucocorticoid and androgenic pathways, respectively. The characterisation of this complex enzyme was initially hampered due to low level in vivo expression of CYP17 Heterologous expression systems have contributed greatly to our current knowledge of CYP17's dual catalytic activity. However, due to the hydrophobic nature of this membrane-bound protein, primarily truncated and modified forms of CYP17 are currently being expressed heterologously. Although the N-terminally modified enzyme has been well characterised, protein structure and function studies still necessitate the expression of unmodified, wild-type CYP17. We report here the expression of a catalytically active, unmodified human CYP17 in the industrial methylotrophic yeast, Pichia pastoris. A typical P450 carbon monoxide difference spectrum, with an absorption maximum at 448 nm and a substrate-induced type I spectrum were recorded using a detergent-solubilised cellular fraction containing CYP17 The expressed enzyme catalysed the conversion of progesterone to 17-hydroxyprogesterone as well as 16-hydroxyprogesterone, a product unique to human and chimpanzee CYP17 This is the first report showing the heterologous expression of a fully functional human steroidogenic cytochrome P450 enzyme in P. pastoris. (c) 2007 Elsevier B.V. All rights reserved.