4-diphosphocytidyl-2-C-methyl-D-erythritol

• 分子结构分类: 有机化合物 - 核苷、核苷酸和类似物 - 嘧啶核苷酸
• 英文名称: 4-diphosphocytidyl-2-C-methyl-D-erythritol
• 英文别名: CDP-ME；4-CDP-2-C-methyl-D-erythritol(2-)；[[(2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-oxidophosphoryl] [(2R,3S)-2,3,4-trihydroxy-3-methylbutyl] phosphate
• 化学式: C₁₄H₂₃N₃O₁₄P₂
• 分子量: 519.296
• InChiKey: YFAUKWZNPVBCFF-XHIBXCGHSA-L

计算性质

• 辛醇/水分配系数(LogP)：
  -5.9
• 重原子数：
  33
• 可旋转键数：
  11
• 环数：
  2.0
• sp3杂化的碳原子比例：
  0.71
• 拓扑面积：
  277
• 氢给体数：
  6
• 氢受体数：
  14

反应信息

• 作为反应物：
  描述：

  5’-三磷酸腺苷 、 4-diphosphocytidyl-2-C-methyl-D-erythritol 在 magnesium chloride 作用下， 生成 CDP-ME2P
  参考文献：
  名称：

  Expression and Characterization of Soluble 4-Diphosphocytidyl-2-C-methyl-D-erythritol Kinase from Bacterial Pathogens

  摘要：

  Many bacterial pathogens utilize the 2-C-methyl-D-erythritol 4-phosphate pathway for biosynthesizing isoprenoid precursors, a pathway that is vital for bacterial survival and absent from human cells, providing a potential source of drug targets. However, the characterization of 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) kinase (IspE) has been hindered due to a lack of enantiopure CDP-ME and difficulty in obtaining pure IspE. Here, enantiopure CDP-ME was chemically synthesized and recombinant IspE from bacterial pathogens were purified and characterized. Although gene disruption was not possible in Mycobacterium tuberculosis, IspE is essential in Mycobacterium smegmatis. The biochemical and kinetic characteristics of IspE provide the basis for development of a high throughput screen and structural characterization.

  DOI：

  10.1016/j.chembiol.2009.10.014
• 作为产物：
  描述：

  2-C-methyl-D-erythritol 4-phosphate 、 cytidine 5'-monophosphate 在 N-甲基咪唑 、 N,N-二甲基苯胺 、 三乙胺 、 三氟乙酸酐 作用下， 以 乙腈 为溶剂， 反应 2.0h， 以45%的产率得到4-diphosphocytidyl-2-C-methyl-D-erythritol
  参考文献：
  名称：

  Expression and Characterization of Soluble 4-Diphosphocytidyl-2-C-methyl-D-erythritol Kinase from Bacterial Pathogens

  摘要：

  Many bacterial pathogens utilize the 2-C-methyl-D-erythritol 4-phosphate pathway for biosynthesizing isoprenoid precursors, a pathway that is vital for bacterial survival and absent from human cells, providing a potential source of drug targets. However, the characterization of 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) kinase (IspE) has been hindered due to a lack of enantiopure CDP-ME and difficulty in obtaining pure IspE. Here, enantiopure CDP-ME was chemically synthesized and recombinant IspE from bacterial pathogens were purified and characterized. Although gene disruption was not possible in Mycobacterium tuberculosis, IspE is essential in Mycobacterium smegmatis. The biochemical and kinetic characteristics of IspE provide the basis for development of a high throughput screen and structural characterization.

  DOI：

  10.1016/j.chembiol.2009.10.014

文献信息

• Biosynthesis of terpenoids: YgbB protein converts 4-diphosphocytidyl-2C-methyl- <scp>d</scp> -erythritol 2-phosphate to 2C-methyl- <scp>d</scp> -erythritol 2,4-cyclodiphosphate
  作者：Stefan Herz、Juraithip Wungsintaweekul、Christoph A. Schuhr、Stefan Hecht、Holger Lüttgen、Sylvia Sagner、Monika Fellermeier、Wolfgang Eisenreich、Meinhart H. Zenk、Adelbert Bacher、Felix Rohdich

  DOI：10.1073/pnas.040554697

  日期：2000.3.14

  In many microorganisms, the putative orthologs of the Escherichia coli ygbB gene are tightly linked or fused to putative orthologs of ygbP , which has been shown earlier to be involved in terpenoid biosynthesis. The ygbB gene of E. coli was expressed in a recombinant E. coli strain and was shown to direct the synthesis of a soluble, 17-kDa polypeptide. The recombinant protein was found to convert 4-diphosphocytidyl-2C-methyl- d -erythritol 2-phosphate into 2C-methyl- d -erythritol 2,4-cyclodiphosphate and CMP. The structure of the reaction product was established by NMR spectroscopy using ¹³ C-labeled substrate samples. The enzyme-catalyzed reaction requires Mn ²⁺ or Mg ²⁺ but no other cofactors. Radioactivity from [2- ¹⁴ C]2C-methyl- d -erythritol 2,4-cyclodiphosphate was diverted efficiently to carotenoids by isolated chromoplasts from Capsicum annuum and, thus, was established as an intermediate in the deoxyxylulose phosphate pathway of isoprenoid biosynthesis. YgbB protein also was found to convert 4-diphosphocytidyl-2C-methyl- d -erythritol into 2C-methyl- d -erythritol 3,4-cyclophosphate. This compound does not serve as substrate for the formation of carotenoids by isolated chromoplasts and is assumed to be an in vitro product without metabolic relevance.

  在许多微生物中，Escherichia coli的ygbB基因的推定同源物通常与ygbP的推定同源物紧密链接或融合在一起，而ygbP早已被证明参与萜类化合物的生物合成。大肠杆菌的ygbB基因在重组的大肠杆菌菌株中表达，并被证明可以指导合成一种可溶性的17 kDa多肽。发现重组蛋白质可以将4-二磷酸胞苷基-2C-甲基-d-赤藓糖醇2-磷酸转化为2C-甲基-d-赤藓糖醇2,4-环二磷酸和CMP。使用13C标记的底物样品，通过NMR光谱法确定了反应产物的结构。酶催化的反应需要Mn2+或Mg2+，但不需要其他辅因子。[2-14C]2C-甲基-d-赤藓糖醇2,4-环二磷酸的放射性能有效地被辣椒素细胞色素体转化为类胡萝卜素，并因此被确定为异戊烯类化合物生物合成的去氧木糖磷酸途径的中间体。发现YgbB蛋白还可以将4-二磷酸胞苷基-2C-甲基-d-赤藓糖醇转化为2C-甲基-d-赤藓糖醇3,4-环磷酸。这种化合物不能作为细胞色素体形成类胡萝卜素的底物，被认为是一种无代谢相关的体外产物。
• Characterization of the <i>Mycobacterium tuberculosis</i> 4-Diphosphocytidyl-2- <i>C</i> -Methyl- <scp>d</scp> -Erythritol Synthase: Potential for Drug Development
  作者：Hyungjin Eoh、Amanda C. Brown、Lori Buetow、William N. Hunter、Tanya Parish、Devinder Kaur、Patrick J. Brennan、Dean C. Crick

  DOI：10.1128/jb.00925-07

  日期：2007.12.15

  Mycobacterium tuberculosis utilizes the methylerythritol phosphate (MEP) pathway for biosynthesis of isopentenyl diphosphate and its isomer, dimethylallyl diphosphate, precursors of all isoprenoid compounds. This pathway is of interest as a source of new drug targets, as it is absent from humans and disruption of the responsible genes has shown a lethal phenotype for Escherichia coli . In the MEP pathway, 4-diphosphocytidyl-2- C -methyl- d -erythritol is formed from 2- C -methyl- d -erythritol 4-phosphate (MEP) and CTP in a reaction catalyzed by a 4-diphosphocytidyl-2- C -methyl- d -erythritol synthase (IspD). In the present work, we demonstrate that Rv3582c is essential for M. tuberculosis : Rv3582c has been cloned and expressed, and the encoded protein has been purified. The purified M. tuberculosis IspD protein was capable of catalyzing the formation of 4-diphosphocytidyl-2- C -methyl- d -erythritol in the presence of MEP and CTP. The enzyme was active over a broad pH range (pH 6.0 to 9.0), with peak activity at pH 8.0. The activity was absolutely dependent upon divalent cations, with 20 mM Mg ²⁺ being optimal, and replacement of CTP with other nucleotide 5′-triphosphates did not support activity. Under the conditions tested, M. tuberculosis IspD had K _m values of 58.5 μM for MEP and 53.2 μM for CTP. Calculated k _cat and k _cat / K _m values were 0.72 min ⁻¹ and 12.3 mM ⁻¹ min ⁻¹ for MEP and 1.0 min ⁻¹ and 18.8 mM ⁻¹ min ⁻¹ for CTP, respectively.

  摘要 结核分枝杆菌 结核分枝杆菌利用赤藓醇磷酸甲酯（MEP）途径进行二磷酸异戊烯酯及其异构体二磷酸二甲烯丙酯（所有异戊烯化合物的前体）的生物合成。这一途径是新药靶点的重要来源，因为人类不存在这一途径，而破坏相关基因会导致大肠杆菌出现致死表型。 大肠杆菌 .在 MEP 途径中，4-二磷酸胞嘧啶-2-C C -甲基- d -赤藓醇由 2- C -甲基- d -4-磷酸赤藓醇（MEP）和CTP在4-二磷酸-2- C -甲基- d -赤藓糖醇合成酶（IspD）催化的反应。在本研究中，我们证明了 Rv3582c 对结核杆菌的生长至关重要。 结核杆菌 Rv3582c：Rv3582c 已被克隆和表达，其编码蛋白已被纯化。纯化的 结核杆菌 IspD 蛋白能够催化形成 4-二磷酸胞嘧啶-2-C C -甲基- d -赤藓醇。该酶在广泛的 pH 值范围（pH 值 6.0 至 9.0）内都具有活性，在 pH 值 8.0 时活性达到峰值。该酶的活性完全依赖于二价阳离子，20 mM Mg 2+ 用其他核苷酸 5′-三磷酸酯代替 CTP 并不支持其活性。在测试条件下 结核杆菌 IspD 具有 K m 为 58.5 μM，CTP 为 53.2 μM。计算得出 k cat 和 k cat / K m 值为 0.72 min -1 和 12.3 毫摩尔 -1 分钟 -1 和 12.3 毫摩尔 -1 和 18.8 毫摩尔 -1 分钟 -1 和 18.8 mM -1 min -1。
• Biosynthesis of Isoprenoids: Characterization of a Functionally Active Recombinant 2-C-methyl-D-erythritol 4-phosphate Cytidyltransferase (IspD) from Mycobacterium tuberculosis H37Rv
  作者：Wenjun Shi、Jianfang Feng、Min Zhang、Xuhui Lai、Shengfeng Xu、Xuelian Zhang、Honghai Wang

  DOI：10.5483/bmbrep.2007.40.6.911

  日期：2007.11.30

  Tuberculosis, caused by Mycobacterium tuberculosis, continues to be one of the leading infectious diseases to humans. It is urgent to discover novel drug targets for the development of antitubercular agents. The 2-C-methyl-Derythritol-4-phosphate (MEP) pathway for isoprenoid biosynthesis has been considered as an attractive target for the discovery of novel antibiotics for its essentiality in bacteria and absence in mammals. MEP cytidyltransferase (IspD), the third-step enzyme of the pathway, catalyzes MEP and CTP to form 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME) and PPi. In the work, ispD gene from M. tuberculosis H37Rv (MtIspD) was cloned and expressed. With N-terminal fusion of a histidine-tagged sequence, MtIspD could be purified to homogeneity by one-step nickel affinity chromatography. MtIspD exists as a homodimer with an apparent molecular mass of 52 kDa. Enzyme property analysis revealed that MtIspD has high specificity for pyrimidine bases and narrow divalent cation requirements, with maximal activity found in the presence of CTP and $Mg^2+}$. The turnover number of MtIspD is $3.4 s^-1}$. The Km for MEP and CTP are 43 and $92\mu}M$, respectively. Furthermore, MtIspD shows thermal instable above $50^\circ}C$. Circular dichroism spectra revealed that the alteration of tertiary conformation is closely related with sharp loss of enzyme activity at higher temperature. This study is expected to help better understand the features of IspD and provide useful information for the development of novel antibiotics to treat M. tuberculosis.

  由结核分枝杆菌引起的结核病仍然是人类的主要传染病之一。发现新的药物靶点以开发抗结核药物迫在眉睫。异戊二烯生物合成的 2-C-甲基赤藓糖醇-4-磷酸（MEP）途径在细菌中必不可少，但在哺乳动物中却不存在，因此被认为是发现新型抗生素的一个有吸引力的靶点。MEP 细胞基转移酶（IspD）是该途径的第三步酶，催化 MEP 和 CTP 形成 4-二磷酸胞苷基-2-C-甲基赤藓醇（CDP-ME）和 PPi。在这项工作中，克隆并表达了结核杆菌 H37Rv 的 ispD 基因（MtIspD）。通过组氨酸标记序列的 N 端融合，MtIspD 可通过一步镍亲和层析纯化至均一。MtIspD 以同源二聚体形式存在，表观分子质量为 52 kDa。酶特性分析表明，MtIspD 对嘧啶碱基具有高度特异性，对二价阳离子的要求很低，在 CTP 和 $Mg^2+}$ 存在时活性最大。MtIspD 的周转次数为 $3.4 s^-1}$。MEP 和 CTP 的 Km 分别为 43 和 $92\mu}M$ 。此外，MtIspD 在 $50^\circ}C$ 以上显示出热不稳定性。圆二色光谱显示，三级构象的改变与酶活性在较高温度下急剧下降密切相关。这项研究有望帮助人们更好地理解IspD的特征，并为开发治疗结核杆菌的新型抗生素提供有用信息。
• Biosynthesis of terpenoids: YchB protein of <i>Escherichia coli</i> phosphorylates the 2-hydroxy group of 4-diphosphocytidyl-2C-methyl- <scp>d</scp> -erythritol
  作者：Holger Lüttgen、Felix Rohdich、Stefan Herz、Juraithip Wungsintaweekul、Stefan Hecht、Christoph A. Schuhr、Monika Fellermeier、Sylvia Sagner、Meinhart H. Zenk、Adelbert Bacher、Wolfgang Eisenreich

  DOI：10.1073/pnas.97.3.1062

  日期：2000.2

  A comparative analysis of all published complete genomes indicated that the putative orthologs of the unannotated ychB gene of Escherichia coli follow the distribution of the dxs , dxr , and ygbP genes, which have been shown to specify enzymes of the deoxyxylulose phosphate pathway of terpenoid biosynthesis, thus suggesting that the hypothetical YchB protein also is involved in that pathway. To test this hypothesis, the E. coli ychB gene was expressed in a homologous host. The recombinant protein was purified to homogeneity and was shown to phosphorylate 4-diphosphocytidyl-2C-methyl- d -erythritol in an ATP-dependent reaction. The reaction product was identified as 4-diphosphocytidyl-2C-methyl- d -erythritol 2-phosphate by NMR experiments with various ¹³ C-labeled substrate samples. A ¹⁴ C-labeled specimen of this compound was converted efficiently into carotenoids by isolated chromoplasts of Capsicum annuum . The sequence of E. coli YchB protein is similar to that of the protein predicted by the tomato cDNA pTOM41 (30% identity), which had been implicated in the conversion of chloroplasts to chromoplasts.

  对所有已发表的完整基因组进行比较分析表明，大肠杆菌未注释的ychB基因的推测同源基因遵循了dxs、dxr和ygbP基因的分布，这些基因已被证明是萜类生物合成中脱氧木糖磷酸途径酶的指定基因，因此暗示假设的YchB蛋白也参与了该途径。为了验证这一假设，将大肠杆菌ychB基因在同源宿主中表达。重组蛋白被纯化至同质性，并显示出在ATP依赖性反应中磷酸化4-二磷酸胞苷-2C-甲基-d-赤藓糖。通过使用各种13C标记底物样品的NMR实验，将反应产物鉴定为4-二磷酸胞苷-2C-甲基-d-赤藓糖2-磷酸酯。这种化合物的14C标记标本被分离的辣椒色素体高效地转化为类胡萝卜素。大肠杆菌YchB蛋白的序列与番茄cDNA pTOM41预测的蛋白的序列相似（30%的同源性），后者已被认为参与了叶绿体向色素体的转化。
• Chemoenzymatic synthesis of 4-diphosphocytidyl-2-C-methyl-d-erythritol: a substrate for IspE
  作者：Prabagaran Narayanasamy、Hyungjin Eoh、Dean C. Crick

  DOI：10.1016/j.tetlet.2008.05.074

  日期：2008.7

  Enantiomerically pure 2-C-methyl-D-erythritol 4-phosphate 1 (MEP) is synthesized from 1,2-O-isopropylidene-alpha-D-xylofuranose via facile benzylation in good yield. Subsequently, 1 is used for enzymatic synthesis of 4-diphosphocytidyl-2-C-methyl-D-erythritol 2 (CDP-ME) using 4-diphosphocytidyl-2-Cmethyl-D-erythritol synthase (IspD). The chemoenzymatically synthesized 2 can be used as substrate for assay of IspE and for high throughput screening to identify 1spE inhibitors. (c) 2008 Elsevier Ltd. All rights reserved.