uridine 5'-diphospho-α-D-galactofuranose

• 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: uridine 5'-diphospho-α-D-galactofuranose
• 英文别名: UDP-alpha-D-galactofuranose(2-)；[(2R,3R,4R,5S)-5-[(1R)-1,2-dihydroxyethyl]-3,4-dihydroxyoxolan-2-yl] [[(2R,3S,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-oxidophosphoryl] phosphate
• 化学式: C₁₅H₂₂N₂O₁₇P₂
• 分子量: 564.29
• InChiKey: ZQLQOXLUCGXKHS-SIAUPFDVSA-L

计算性质

• 辛醇/水分配系数(LogP)：
  -6.8
• 重原子数：
  36
• 可旋转键数：
  10
• 环数：
  3.0
• sp3杂化的碳原子比例：
  0.73
• 拓扑面积：
  297
• 氢给体数：
  7
• 氢受体数：
  17

反应信息

• 作为反应物：
  描述：

  uridine 5'-diphospho-α-D-galactofuranose 在 uridine 5′-diphosphate-galactopyranose mutase 作用下， 生成 uridine 5'-(α-D-galactopyranosyl diphosphate)
  参考文献：
  名称：

  Virtual Screening for UDP-Galactopyranose Mutase Ligands Identifies a New Class of Antimycobacterial Agents

  摘要：

  Galactofuranose (Gay) is present in glycans critical for the virulence and viability of several pathogenic microbes, including Mycobacterium tuberculosis, yet the monosaccharide is absent from mammalian glycans. Uridine 5'-diphosphate-galactopyranose mutase (UGM) catalyzes the formation of UDP-Galf, which is required to produce Galf-containing glycoconjugates. Inhibitors of UGM have therefore been sought, both as antimicrobial leads and as tools to delineate the roles of Galf in cells. Obtaining cell permeable UGM probes by either design or high throughput screens has been difficult, as has elucidating how UGM binds small molecule, noncarbohydrate inhibitors. To address these issues, we employed structure-based virtual screening to uncover new inhibitor chemotypes, including a triazolothiadiazine series. These compounds are among the most potent antimycobacterial UGM inhibitors described. They also facilitated determination of a UGM small molecule inhibitor structure, which can guide optimization. A comparison of results from the computational screen and a high-throughput fluorescence polarization (FP) screen indicated that the scaffold hits from the former had been evaluated in the FP screen but missed. By focusing on promising compounds, the virtual screen rescued false negatives, providing a blueprint for generating new UGM probes and therapeutic leads.

  DOI：

  10.1021/acschembio.5b00370
• 作为产物：
  描述：

  uridine 5'-(α-D-galactopyranosyl diphosphate) 生成 uridine 5'-diphospho-α-D-galactofuranose
  参考文献：
  名称：

  Galactofuranose biosynthesis in Escherichia coli K-12: identification and cloning of UDP-galactopyranose mutase

  摘要：

  我们从大肠杆菌K-12 rfb区克隆了两个开放阅读框架（orf6和orf8）。这些基因在T7lac启动子的控制下在大肠杆菌中表达，产生大量的重组蛋白，其中大部分积累在不溶性包涵体中。然而，获得了足够的可溶性蛋白用于放射性测定，该测定旨在检测UDP-半乳糖吡喃糖异构酶活性（将UDP-半乳糖吡喃糖转化为UDP-半乳糖呋喃糖）。该测定基于高压液相色谱法分离糖磷酸，这些糖磷酸来自通过磷酸二酯酶处理两种形式的UDP-半乳糖。粗制orf6基因产物将UDP- [α-D-U-14C] -半乳糖吡喃糖转化为一种化合物，该化合物在磷酸二酯酶处理后产生一个保留时间与合成α-半乳糖呋喃糖-1-磷酸相同的化合物。从不含orf6表达质粒或orf8表达细胞的提取物中未检测到任何异构酶活性。orf6基因产物通过阴离子交换色谱和疏水相互作用色谱纯化。粗提取物和纯化蛋白均将6-9％的UDP-半乳糖吡喃糖转化为呋喃糖形式。该酶也被证明具有催化反应的能力；在这种情况下，观察到约86％的呋喃糖转化为吡喃糖。这些观察结果强烈表明orf6编码UDP-半乳糖吡喃糖异构酶（EC 5.4.99.9），我们建议将该基因命名为glf。纯化的UDP-半乳糖吡喃糖异构酶的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示出一个主要带，电喷雾质谱分析表明分子量为42,960 +/- 8的单一主要物种，与Glf蛋白计算的相符。N末端测序显示重组蛋白的前15个氨基酸与已发表序列相对应。纯化的重组酶的UV-可见光谱表明该蛋白质含有一种黄酮辅因子，我们已鉴定为黄酮腺嘌呤二核苷酸。

  DOI：

  10.1128/jb.178.4.1047-1052.1996

文献信息

• Expression, Purification, and Characterization of a Galactofuranosyltransferase Involved in <i>Mycobacterium t</i><i>uberculosis</i> Arabinogalactan Biosynthesis
  作者：Natisha L. Rose、Gladys C. Completo、Shuang-Jun Lin、Michael McNeil、Monica M. Palcic、Todd L. Lowary

  DOI：10.1021/ja058254d

  日期：2006.5.1

  beta-(1-->6) linkages. A single enzyme, glfT, has been shown to synthesize both glycosidic linkages. We report here the first high-level expression and purification of glfT by expression of the Rv3808c gene in Escherichia coli C41(DE3). Following a three-step purification procedure, 3-7 mg of protein of >95% purity was isolated from each liter of culture. We subsequently probed the substrate specificity

  结核分枝杆菌细胞壁的主要结构成分是脂化多糖，即霉菌酰-阿拉伯半乳聚糖-肽聚糖 (mAGP) 复合物。这种糖缀合物在生物体的生存中起着关键作用，因此，参与其生物合成的酶作为药物作用位点受到了关注。mAGP 的核心是由 D-呋喃半乳糖残基组成的半乳聚糖，通过交替的 β-(1-->5) 和 β-(1-->6) 键连接。一种单一的酶 glfT 已被证明可以合成两种糖苷键。我们在这里报告了通过在大肠杆菌 C41(DE3) 中表达 Rv3808c 基因来首次高水平表达和纯化 glfT。遵循三步纯化程序，从每升培养物中分离出 3-7 mg 纯度 > 95% 的蛋白质。我们随后通过评估一组潜在的单糖和寡糖底物探索了 glfT 的底物特异性，并首次证明三糖是比二糖更好的底物，并且一种双糖，其中末端 D-呋喃半乳糖残基被替换为L-阿拉伯呋喃糖部分是弱底物。使用四个寡糖受体酶的动力学表征给出了从 204 microM
• Galactofuranose biosynthesis in Escherichia coli K-12: identification and cloning of UDP-galactopyranose mutase
  作者：P M Nassau、S L Martin、R E Brown、A Weston、D Monsey、M R McNeil、K Duncan

  DOI：10.1128/jb.178.4.1047-1052.1996

  日期：1996.2

  We have cloned two open reading frames (orf6 and orf8) from the Escherichia coli K-12 rfb region. The genes were expressed in E. coli under control of the T7lac promoter, producing large quantities of recombinant protein, most of which accumulated in insoluble inclusion bodies. Sufficient soluble protein was obtained, however, for use in a radiometric assay designed to detect UDP-galactopyranose mutase activity (the conversion of UDP-galactopyranose to UDP-galactofuranose). The assay is based upon high-pressure liquid chromatography separation of sugar phosphates released from both forms of UDP-galactose by phosphodiesterase treatment. The crude orf6 gene product converted UDP-[alpha-D-U-14C]-galactopyranose to a product which upon phosphodiesterase treatment gave a compound with a retention time identical to that of synthetic alpha-galactofuranose-1-phosphate. No mutase activity was detected in extracts from cells lacking the orf6 expression plasmid or from orf8-expressing cells. The orf6 gene product was purified by anion-exchange chromatography and hydrophobic interaction chromatography. Both the crude extract and the purified protein converted 6 to 9% of the UDP-galactopyranose to the furanose form. The enzyme was also shown to catalyze the reverse reaction; in this case an approximately 86% furanose-to-pyranose conversion was observed. These observations strongly suggest that orf6 encodes UDP-galactopyranose mutase (EC 5.4.99.9), and we propose that the gene be designated glf accordingly. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified UDP-galactopyranose mutase revealed one major band, and analysis by electrospray mass spectrometry indicated a single major species with a molecular weight of 42,960 +/- 8, in accordance with that calculated for the Glf protein. N-terminal sequencing revealed that the first 15 amino acids of the recombinant protein corresponded to those expected from the published sequence. UV-visible spectra of purified recombinant enzyme indicated that the protein contains a flavin cofactor, which we have identified as flavin adenine dinucleotide.

  我们从大肠杆菌K-12 rfb区克隆了两个开放阅读框架（orf6和orf8）。这些基因在T7lac启动子的控制下在大肠杆菌中表达，产生大量的重组蛋白，其中大部分积累在不溶性包涵体中。然而，获得了足够的可溶性蛋白用于放射性测定，该测定旨在检测UDP-半乳糖吡喃糖异构酶活性（将UDP-半乳糖吡喃糖转化为UDP-半乳糖呋喃糖）。该测定基于高压液相色谱法分离糖磷酸，这些糖磷酸来自通过磷酸二酯酶处理两种形式的UDP-半乳糖。粗制orf6基因产物将UDP- [α-D-U-14C] -半乳糖吡喃糖转化为一种化合物，该化合物在磷酸二酯酶处理后产生一个保留时间与合成α-半乳糖呋喃糖-1-磷酸相同的化合物。从不含orf6表达质粒或orf8表达细胞的提取物中未检测到任何异构酶活性。orf6基因产物通过阴离子交换色谱和疏水相互作用色谱纯化。粗提取物和纯化蛋白均将6-9％的UDP-半乳糖吡喃糖转化为呋喃糖形式。该酶也被证明具有催化反应的能力；在这种情况下，观察到约86％的呋喃糖转化为吡喃糖。这些观察结果强烈表明orf6编码UDP-半乳糖吡喃糖异构酶（EC 5.4.99.9），我们建议将该基因命名为glf。纯化的UDP-半乳糖吡喃糖异构酶的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示出一个主要带，电喷雾质谱分析表明分子量为42,960 +/- 8的单一主要物种，与Glf蛋白计算的相符。N末端测序显示重组蛋白的前15个氨基酸与已发表序列相对应。纯化的重组酶的UV-可见光谱表明该蛋白质含有一种黄酮辅因子，我们已鉴定为黄酮腺嘌呤二核苷酸。
• Identification of a Novel Galactosyl Transferase Involved in Biosynthesis of the Mycobacterial Cell Wall
  作者：Katarína Mikušová、Martina Beláňová、Jana Korduláková、Kristine Honda、Michael R. McNeil、Sebabrata Mahapatra、Dean C. Crick、Patrick J. Brennan

  DOI：10.1128/jb.00489-06

  日期：2006.9.15

  The possibility of the Rv3782 protein of Mycobacterium tuberculosis being a putative galactosyl transferase (GalTr) implicated in galactan synthesis arose from its similarity to the known GalTr Rv3808c, its classification as a nucleotide sugar-requiring inverting glycosyltransferase (GT-2 family), and its location within the “possible arabinogalactan biosynthetic gene cluster” of M. tuberculosis. In order to study the function of the enzyme, active membrane and cell wall fractions from Mycobacterium smegmatis containing the overexpressed Rv3782 protein were incubated with endogenous decaprenyldiphosphoryl- N -acetylglucosaminyl-rhamnose (C ₅₀ -P-P-GlcNAc-Rha) as the primary substrate for galactan synthesis and UDP-[ ¹⁴ C]galactopyranose as the immediate precursor of UDP-[ ¹⁴ C]galactofuranose, the ultimate source of all of the galactofuranose (Gal f ) units of galactan. Obvious increased and selective synthesis of C ₅₀ -P-P-GlcNAc-Rha-Gal f- Gal f , the earliest product in the pathway leading to the fully polymerized galactan, was observed, suggesting that Rv3782 encodes a GalTr involved in the first stages of galactan synthesis. Time course experiments pointed to a possible bifunctional enzyme responsible for the initial synthesis of C ₅₀ -P-P-GlcNAc-Rha-Gal f , followed by immediate conversion to C ₅₀ -P-P-GlcNAc-Rha-Gal f- Gal f . Thus, Rv3782 appears to be the initiator of galactan synthesis, while Rv3808c continues with the subsequent polymerization events.

  摘要 结核分枝杆菌 Rv3782 蛋白的可能性 结核分枝杆菌 Rv3782 蛋白与已知的半乳糖基转移酶 Rv3808c 相似，被归类为核苷酸糖要求倒置糖基转移酶（GT-2 家族），并且位于结核分枝杆菌的 "可能的阿拉伯半乳聚糖生物合成基因簇 "内，因此有可能是一种与半乳聚糖合成有关的假定半乳糖基转移酶（GalTr）。 结核杆菌的 "可能的阿拉伯半乳聚糖生物合成基因簇 "中。 为了研究该酶的功能，研究人员从烟草分枝杆菌（Mycobacterium smegmat）中提取了活性膜和细胞壁组分。 分枝杆菌 为了研究该酶的功能，将含有过表达 Rv3782 蛋白的烟曲霉的活性膜和细胞壁分馏物与内源性十烯丙基二磷酸- N N -乙酰葡糖胺酰鼠李糖（C 50 -P-GlcNAc-Rha）作为半乳糖合成的主要底物与 UDP-[... 14 C]吡喃半乳糖的直接前体。 14 C]半乳糖呋喃糖是所有半乳糖呋喃糖（Gal f ）半乳糖单位的最终来源。C 50 -P-GlcNAc-Rha-Gal f- Gal f 的最早产物，表明 Rv3782 编码的 GalTr 参与了半乳糖合成的最初阶段。时间进程实验表明，一种可能的双功能酶负责 C 50 -P-P-GlcNAc-Rha-Gal f 随后立即转化为 C 50 -P-P-GlcNAc-Rha-Gal f- Gal f .因此，Rv3782 似乎是半乳糖合成的启动器，而 Rv3808c 则继续随后的聚合过程。
• Galactosyl Transferases in Mycobacterial Cell Wall Synthesis
  作者：Martina Beláňová、Petronela Dianišková、Patrick J. Brennan、Gladys C. Completo、Natisha L. Rose、Todd L. Lowary、Katarína Mikušová

  DOI：10.1128/jb.01326-07

  日期：2008.2

  Two galactosyl transferases can apparently account for the full biosynthesis of the cell wall galactan of mycobacteria. Evidence is presented based on enzymatic incubations with purified natural and synthetic galactofuranose (Galf) acceptors that the recombinant galactofuranosyl transferase, GlfT1, from Mycobacterium smegmatis, the Mycobacterium tuberculosis Rv3782 ortholog known to be involved in

  两种半乳糖基转移酶显然可以解释分枝杆菌细胞壁半乳聚糖的完整生物合成。证据是基于与纯化的天然和合成呋喃半乳糖 (Galf) 受体的酶促孵育，来自耻垢分枝杆菌的重组呋喃半乳糖基转移酶 GlfT1，已知参与半乳聚糖形成的初始步骤的结核分枝杆菌 Rv3782 直向同源物，具有双重β- (1-->4) 和 β-(1-->5) Galf 转移酶活性，并且该酶的产物 decaprenyl-PP-GlcNAc-Rha-Galf-Galf 作为直接底物用于催化完全聚合另一种双功能 Galf 转移酶 GlfT2，即 Rv3808c 酶。
• A tethering mechanism for length control in a processive carbohydrate polymerization
  作者：John F. May、Rebecca A. Splain、Christine Brotschi、Laura L. Kiessling

  DOI：10.1073/pnas.0901407106

  日期：2009.7.21

  Carbohydrate polymers are the most abundant organic substances on earth. Their degrees of polymerization range from tens to thousands of units, yet polymerases generate the relevant lengths without the aid of a template. To gain insight into template-independent length control, we investigated how the mycobacterial galactofuranosyltransferase GlfT2 mediates formation of the galactan, a polymer of galactofuranose residues that is an integral part of the cell wall. We show that isolated recombinant GlfT2 can catalyze the synthesis of polymers with degrees of polymerization that are commensurate with values observed in mycobacteria, indicating that length control by GlfT2 is intrinsic. Investigations using synthetic substrates reveal that GlfT2 is processive. The data indicate that GlfT2 controls length by using a substrate tether, which is distal from the site of elongation. The strength of interaction of that tether with the polymerase influences the length of the resultant polymer. Thus, our data identify a mechanism for length control by a template-independent polymerase.

  碳水化合物聚合物是地球上最丰富的有机物质。它们的聚合度从几十到数千个单位不等，但聚合酶可以在没有模板的情况下生成相关长度。为了了解模板无关的长度控制，我们研究了结核分枝杆菌半乳糖酰基转移酶GlfT2如何介导半乳糖酸聚合物（一种由半乳糖酸残基组成的聚合物，是细胞壁的组成部分）的形成。我们展示了独立的重组GlfT2可以催化合成与结核分枝杆菌中观察到的聚合度相一致的聚合物，表明GlfT2的长度控制是内在的。使用合成底物的调查表明GlfT2是过程性的。数据表明，GlfT2通过使用远离延伸位点的底物系留来控制长度。该系留与聚合酶的相互作用强度影响所得聚合物的长度。因此，我们的数据确定了模板无关聚合酶长度控制的机制。