3alpha,12alpha-Dihydroxy-7-oxo-5beta-cholanate

• 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: 3alpha,12alpha-Dihydroxy-7-oxo-5beta-cholanate
• 英文别名: (4R)-4-[(3R,5S,8R,9S,10S,12S,13R,14S,17R)-3,12-dihydroxy-10,13-dimethyl-7-oxo-1,2,3,4,5,6,8,9,11,12,14,15,16,17-tetradecahydrocyclopenta[a]phenanthren-17-yl]pentanoate
• 化学式: C24H37O5-
• 分子量: 405.5
• InChiKey: RHCPKKNRWFXMAT-RRWYKFPJSA-M

计算性质

• 辛醇/水分配系数(LogP)：
  3.9
• 重原子数：
  29
• 可旋转键数：
  3
• 环数：
  4.0
• sp3杂化的碳原子比例：
  0.92
• 拓扑面积：
  97.7
• 氢给体数：
  2
• 氢受体数：
  5

反应信息

• 作为产物：
  描述：

  Cholate 、 NADP⁺ 生成 3alpha,12alpha-Dihydroxy-7-oxo-5beta-cholanate 、 氢(+1)阳离子 、 NADPH(4-)
  参考文献：
  名称：

  在寻找可持续的化学过程中：假单胞菌7α-和7β-羟基类固醇脱氢酶的克隆，重组表达和功能表征。

  摘要：

  烟酸梭状芽孢杆菌的烟酰胺腺嘌呤二核苷酸磷酸依赖性7α-羟基类固醇脱氢酶（7α-HSDH）和7β-羟基类固醇脱氢酶（7β-HSDH）催化伯胆汁酸通过7-酮胆汁酸中间体发生差向异构化反应，可能适合用作生物催化具有药理学意义的胆汁酸衍生物的合成。枯草衣原体7α-HSDH已纯化至同质，N端序列已通过Edman测序确定。用简并引物对基因片段进行PCR扩增后，已通过对Absum C. absonum基因组DNA进行测序，克隆了完整的基因（786 nt）。通过对7α-HSDH基因5'末端侧翼的基因组DNA区域进行测序，获得了编码7β-HSDH（783 nt）的序列，这两个基因是连续的，可能是同一操纵子的一部分。插入合适的表达载体后，两种HSDHs已成功在大肠杆菌中以重组形式生产，通过亲和层析纯化，并进行了动力学分析，以确定米氏常数（K（m））和特异性常数（k（cat）/ K） （m））在各种胆汁酸衍生物

  DOI：

  10.1007/s00253-011-3798-x

文献信息

• Cloning and sequencing of the 7 alpha-hydroxysteroid dehydrogenase gene from Escherichia coli HB101 and characterization of the expressed enzyme
  作者：T Yoshimoto、H Higashi、A Kanatani、X S Lin、H Nagai、H Oyama、K Kurazono、D Tsuru

  DOI：10.1128/jb.173.7.2173-2179.1991

  日期：1991.4

  The 7 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.159) gene from Escherichia coli HB101 was cloned and expressed in E. coli DH1. The hybrid plasmid pSD1, with a 2.8-kbp insert of chromosomal DNA at the BamHI site of pBR322, was subcloned into pUC19 to construct plasmid pSD3. The entire nucleotide sequence of an inserted PstI-BamHI fragment of plasmid pSD3 was determined by the dideoxy chain-termination method. Within this sequence, the mature enzyme protein-encoding sequence was found to start at a GTG initiation codon and to comprise 765 bp, as judged by comparison with the protein sequence. The deduced amino acid sequence of the enzyme indicated that the molecular weight is 26,778. The transformant of E. coli DH1 harboring pSD3 with a 1.8-kbp fragment showed about 200-fold-higher enzyme activity than the host. The enzyme was purified by a single chromatography step on DEAE-Toyopearl and obtained as crystals, with an activity yield of 39%. The purified enzyme was homogeneous, as judged by sodium dodecyl sulfate gel electrophoresis. The enzyme was most active at pH 8.5 and stable between pH 8 and 9. The enzyme was NAD+ dependent and had a pI of 4.3. The molecular mass was estimated to be 120 kDa by the gel filtration method and 28 kDa by electrophoresis, indicating that the enzyme exists in a tetrameric form.

  大肠杆菌HB101的7α-羟类固醇脱氢酶（EC 1.1.1.159）基因被克隆并在大肠杆菌DH1中表达。将含有2.8-kbp染色体DNA插入到pBR322的BamHI位点的杂交质粒pSD1亚克隆到pUC19中构建出质粒pSD3。通过dideoxy链终止法确定了质粒pSD3的插入PstI-BamHI片段的整个核苷酸序列。在该序列中，成熟酶蛋白编码序列从GTG启动密码子开始，由765个碱基组成，与蛋白质序列比较得出。酶的推导氨基酸序列表明分子量为26,778。携带1.8-kbp片段的大肠杆菌DH1转化体，其酶活性比宿主高约200倍。酶经DEAE-Toyopearl单一色谱步骤纯化，并以晶体形式获得，活性收率为39％。纯化的酶通过十二烷基硫酸钠凝胶电泳判断为均一。该酶在pH 8.5时最活跃，在pH 8和9之间稳定。该酶依赖于NAD+，其等电点为4.3。通过凝胶过滤法估计分子量为120 kDa，通过电泳法估计为28 kDa，表明该酶以四聚体形式存在。
• Characterization and regulation of the NADP-linked 7 alpha-hydroxysteroid dehydrogenase gene from Clostridium sordellii
  作者：J P Coleman、L L Hudson、M J Adams

  DOI：10.1128/jb.176.16.4865-4874.1994

  日期：1994.8

  A bile acid-inducible NADP-linked 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH) from Clostridium sordellii ATCC 9714 was purified 310-fold by ion-exchange, gel filtration, and dye-ligand affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified enzyme showed one predominant peptide band (30,000 Da). The N-terminal sequence was determined, and the corresponding oligonucleotides were synthesized and used to screen EcoRI and HindIII genomic digests of C. sordellii. Two separate fragments (4,500 bp, EcoRI; 3,200 bp, HindIII) were subsequently cloned by ligation to pUC19 and transformation into Escherichia coli DH5 alpha-MCR. The EcoRI fragment was shown to contain a truncated 7 alpha-HSDH gene, while the HindIII fragment contained the entire coding region. E. coli clones containing the HindIII insert expressed high levels of an NADP-linked 7 alpha-HSDH. Nucleotide sequence analyses suggest that the 7 alpha-HSDH is encoded by a monocistronic transcriptional unit, with DNA sequence elements resembling rho-independent terminators located in both the upstream and downstream flanking regions. The transcriptional start site was located by primer extension analysis. Northern (RNA) blot analysis indicated that induction is mediated at the transcriptional level in response to the presence of bile acid in the growth medium. In addition, growth-phase-dependent expression is observed in uninduced cultures. Analysis of the predicted protein sequence indicates that the enzyme can be classified in the short-chain dehydrogenase group.

  从Clostridium sordellii ATCC 9714中纯化了一种胆汁酸诱导的NADP连接的7α-羟基类固醇脱氢酶（7α-HSDH），经离子交换、凝胶过滤和染料配基亲和层析分离，纯化倍数为310倍。纯化酶的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析显示有一个主要的肽带（30,000 Da）。确定了N端序列，并合成了相应的寡核苷酸，用于筛选C. sordellii的EcoRI和HindIII基因组消化物。随后，将两个独立的片段（4,500 bp，EcoRI；3,200 bp，HindIII）通过连接到pUC19并转化到Escherichia coli DH5 alpha-MCR中进行克隆。EcoRI片段被证明包含一个截短的7α-HSDH基因，而HindIII片段包含整个编码区域。含有HindIII插入物的E. coli克隆表达高水平的NADP连接的7α-HSDH。核苷酸序列分析表明，7α-HSDH由单顺式转录单元编码，上下游侧翼区域均具有类似于rho独立终止子的DNA序列元素。引物延伸分析确定了转录起始位点。Northern（RNA）印迹分析表明，在生长基质中存在胆汁酸的情况下，诱导是通过转录水平介导的。此外，在未诱导的培养物中观察到生长阶段依赖性的表达。预测的蛋白质序列分析表明，该酶可以归类为短链脱氢酶组。
• Cloning and Characterization of the NAD-Dependent 7?-Hydroxysteroid Dehydrogenase from Bacteroides fragilis
  作者：Michael J. Bennett、Susan L. McKnight、James P. Coleman

  DOI：10.1007/s00284-003-4079-4

  日期：2003.12

  The NAD-linked 7alpha-hydroxysteroid dehydrogenase (7-HSDH) from Bacteroides fragilis ATCC 25285 was characterized and its gene cloned. The enzyme displayed optimal activities at pH 8.5 (NAD reduction) and 6.5 (NADH oxidation). The lowest K(m) and highest V(max) values were observed with chenodeoxycholic acid and its conjugates. The protein had subunits of 27.4 kDa and a native size of 110 kDa, suggesting a homotetrameric composition. The enzyme was relatively thermostable, retaining 95% of initial activity after 1 h at 65 degrees C. A DNA probe based on the N-terminal amino acid sequence hybridized to a 2373-bp HindIII fragment of B. fragilis DNA. This fragment was cloned into E. coli and sequenced, revealing a 780-bp open reading frame. The predicted amino acid sequence of the ORF showed strong sequence similarity to three other bacterial 7-HSDHs, all in the short-chain dehydrogenase family. The regulation of expression of this gene is currently under investigation.
• In search of sustainable chemical processes: cloning, recombinant expression, and functional characterization of the 7α- and 7β-hydroxysteroid dehydrogenases from Clostridium absonum
  作者：Erica Elisa Ferrandi、Giulia Maria Bertolesi、Fausto Polentini、Armando Negri、Sergio Riva、Daniela Monti

  DOI：10.1007/s00253-011-3798-x

  日期：2012.9

  dinucleotide phosphate-dependent 7α-hydroxysteroid dehydrogenase (7α-HSDH) and 7β-hydroxysteroid dehydrogenases (7β-HSDH) from Clostridium absonum catalyze the epimerization of primary bile acids through 7-keto bile acid intermediates and may be suitable as biocatalysts for the synthesis of bile acids derivatives of pharmacological interest. C. absonum 7α-HSDH has been purified to homogeneity and the

  烟酸梭状芽孢杆菌的烟酰胺腺嘌呤二核苷酸磷酸依赖性7α-羟基类固醇脱氢酶（7α-HSDH）和7β-羟基类固醇脱氢酶（7β-HSDH）催化伯胆汁酸通过7-酮胆汁酸中间体发生差向异构化反应，可能适合用作生物催化具有药理学意义的胆汁酸衍生物的合成。枯草衣原体7α-HSDH已纯化至同质，N端序列已通过Edman测序确定。用简并引物对基因片段进行PCR扩增后，已通过对Absum C. absonum基因组DNA进行测序，克隆了完整的基因（786 nt）。通过对7α-HSDH基因5'末端侧翼的基因组DNA区域进行测序，获得了编码7β-HSDH（783 nt）的序列，这两个基因是连续的，可能是同一操纵子的一部分。插入合适的表达载体后，两种HSDHs已成功在大肠杆菌中以重组形式生产，通过亲和层析纯化，并进行了动力学分析，以确定米氏常数（K（m））和特异性常数（k（cat）/ K） （m））在各种胆汁酸衍生物