5a(11a)-dehydrotetracycline

• 分子结构分类: 有机化合物 - 苯丙烷和聚酮 - 四环素类
• 英文名称: 5a(11a)-dehydrotetracycline
• 化学式: C₂₂H₂₂N₂O₈
• 分子量: 442.425
• InChiKey: DUAVZCXHIINBQU-ILGMQVKHSA-N

计算性质

• 辛醇/水分配系数(LogP)：
  -0.78
• 重原子数：
  32.0
• 可旋转键数：
  2.0
• 环数：
  4.0
• sp3杂化的碳原子比例：
  0.36
• 拓扑面积：
  178.46
• 氢给体数：
  5.0
• 氢受体数：
  9.0

反应信息

• 作为反应物：
  描述：

  5a(11a)-dehydrotetracycline 在 7,8-didemethyl-8-hydroxy-5-deazariboflavin 、 dehydrotetracycline reductase 、 F₄₂₀ NADPH oxidoreductase from Archaeoglobus fulgidus 、 还原型辅酶II(NADPH)四钠盐 作用下， 生成 四环素
  参考文献：
  名称：

  酿酒酵母生物合成四环素最后步骤的异源催化

  摘要：

  开发抗生素耐药性细菌感染的治疗方法是全球最优先的公共卫生挑战之一。四环素是最重要的抗生素类别之一，已成为抗生素耐药性的牺牲品，因此需要产生新的类似物。许多四环素类似物已通过全合成和半合成获得，但关键的 C 环四环素类似物仍然无法获得。需要新的方法来解锁对这些类似物的访问，而在易处理的宿主（如酿酒酵母）中进行异源生物合成是一种候选方法。C 环类似物的生物合成可以模拟自然界从无水四环素合成四环素，但存在挑战，包括缺乏独特的辅因子 F 420在共同的异源宿主中。为了实现这一目标，本文描述了在酿酒酵母中从异源表达来自三种细菌宿主的三种酶的无水四环素生物合成四环素：无水四环素羟化酶 OxyS、脱氢四环素还原酶 CtcM 和 F 420还原酶 FNO。这种四环素的生物合成是通过 OxyS 在酿酒酵母中仅进行一个羟基化步骤实现的，尽管其先前被表征为双羟化酶。这种单一的羟基化使我们能够纯化并在结构上表征土

  DOI：

  10.1021/acschembio.1c00259
• 作为产物：
  描述：

  脱水差向四环素 在 氧气 、 anhydrotetracycline hydroxylase 、 还原型辅酶II(NADPH)四钠盐 作用下， 生成 5a(11a)-dehydrotetracycline
  参考文献：
  名称：

  酿酒酵母生物合成四环素最后步骤的异源催化

  摘要：

  开发抗生素耐药性细菌感染的治疗方法是全球最优先的公共卫生挑战之一。四环素是最重要的抗生素类别之一，已成为抗生素耐药性的牺牲品，因此需要产生新的类似物。许多四环素类似物已通过全合成和半合成获得，但关键的 C 环四环素类似物仍然无法获得。需要新的方法来解锁对这些类似物的访问，而在易处理的宿主（如酿酒酵母）中进行异源生物合成是一种候选方法。C 环类似物的生物合成可以模拟自然界从无水四环素合成四环素，但存在挑战，包括缺乏独特的辅因子 F 420在共同的异源宿主中。为了实现这一目标，本文描述了在酿酒酵母中从异源表达来自三种细菌宿主的三种酶的无水四环素生物合成四环素：无水四环素羟化酶 OxyS、脱氢四环素还原酶 CtcM 和 F 420还原酶 FNO。这种四环素的生物合成是通过 OxyS 在酿酒酵母中仅进行一个羟基化步骤实现的，尽管其先前被表征为双羟化酶。这种单一的羟基化使我们能够纯化并在结构上表征土

  DOI：

  10.1021/acschembio.1c00259

文献信息

• [EN] METHODS FOR ENGINEERING THERAPEUTICS AND USES THEREOF<br/>[FR] PROCÉDÉS D'INGÉNIÉRIE D'AGENTS THÉRAPEUTIQUES ET UTILISATIONS ASSOCIÉES
  申请人：UNIV COLUMBIA

  公开号：WO2021092589A1

  公开（公告）日：2021-05-14

  The disclosed subject matter provides for genetically modified cells, e.g, fungal cells, that autonomously generates and/or secretes one or more therapeutic molecules, e.g, therapeutic peptides, therapeutic proteins or small therapeutic molecules, in situ. In certain embodiments, the present disclosure provides genetically-engineered fungal cells that generate and secrete tetracycline and analogues thereof.

  所披露的主题提供了基因修饰细胞，例如真菌细胞，这些细胞自主地产生和/或分泌一种或多种治疗分子，例如治疗肽、治疗蛋白质或小型治疗分子，在原位。在某些实施例中，本公开提供了生成和分泌四环素及其类似物的基因工程真菌细胞。
• Cloning and heterologous expression in Streptomyces lividans of Streptomyces rimosus genes involved in oxytetracycline biosynthesis
  作者：C Binnie、M Warren、M J Butler

  DOI：10.1128/jb.171.2.887-895.1989

  日期：1989.2

  The anhydrotetracycline (ATC) oxygenase enzyme which carries out the conversion of ATC to dehydrotetracycline was purified and the N-terminal amino acid sequence was determined. The sequence displays a significant similarity to that of the p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. This is consistent with the activity of the oxygenase, i.e., addition of a hydroxyl moiety to an aromatic ring structure. Oligonucleotide probes were designed and used to clone the corresponding fragment of chromosomal DNA from Streptomyces rimosus. This DNA fragment was used to screen a cosmid library, allowing the isolation of flanking DNA sequences. Surprisingly, the gene was located within the previously cloned cluster of genes involved in the synthesis of the biosynthetic intermediate ATC and not as had been expected (P. M. Rhodes, N. Winskill, E. J. Friend, and M. Warren, J. Gen. Microbiol. 124:329-338, 1981) at a separate locus on the other side of the chromosome. Subcloning of an appropriate DNA fragment from one of the cosmid clones onto pIJ916 produced Streptomyces lividans transformants which synthesized oxytetracycline.

  无水四环素(ATC)氧化酶酶能够将ATC转化为去氢四环素，并已被纯化，其N末端氨基酸序列已被确定。该序列与假单胞菌荧光素对羟基苯甲酸羟化酶的相似性显著，这与氧化酶的活性一致，即向芳环结构中添加羟基基团。寡核苷酸探针被设计并用于从链霉菌里莫瑟斯的染色体DNA中克隆相应的片段。这个DNA片段被用来筛选cosmid文库，从而分离出相邻的DNA序列。令人惊讶的是，这个基因位于先前克隆的涉及合成生物合成中间体ATC的基因簇内，而不是像预期的那样(P.M. Rhodes, N. Winskill, E.J. Friend和M. Warren，J. Gen. Microbiol. 124:329-338，1981)在染色体的另一侧的一个单独的位点。从一个cosmid克隆体中亚克隆一个适当的DNA片段到pIJ916上，可以产生合成土霉素的链霉亮菌转化体。
• Isolation of pure anhydrotetracycline oxygenase from <i>Streptomyces aureofaciens</i>
  作者：I Vancurová、J Volc、M Flieger、J Neuzil、J Novotná、J Vlach、V Bĕhal

  DOI：10.1042/bj2530263

  日期：1988.7.1

  Anhydrotetracycline oxygenase was purified to homogeneity from Streptomyces aureofaciens, a producer of tetracycline. The enzyme was purified 60-fold in a 40% yield by a two-step procedure using a combination of hydrophobic chromatography and ion-exchange h.p.l.c. Purified anhydrotetracycline oxygenase was homogeneous according to SDS/polyacrylamide-gel electrophoresis, isoelectric focusing, ion-exchange h.p.l.c. on a Mono Q HR 5/5 column and size-exclusion h.p.l.c. on a TSK G 3000 SW column. The enzyme consists of two subunits of Mr 57,500, as determined by SDS/polyacrylamide-gel electrophoresis.

  无水四环素氧化酶从四环素生产菌Streptomyces aureofaciens中纯化至同质性。使用亲水性层析和离子交换高效液相色谱的组合方法，经过两步程序纯化，得到了纯度为60倍的酶，收率为40%。经SDS /聚丙烯酰胺凝胶电泳、等电聚焦、在Mono Q HR 5/5柱上的离子交换高效液相色谱和在TSK G 3000 SW柱上的分子大小排除高效液相色谱，纯化的无水四环素氧化酶是同质的。该酶由两个分子量为57,500的亚基组成，经SDS /聚丙烯酰胺凝胶电泳确定。
• Ablation of the otcC Gene Encoding a Post-polyketide Hydroxylase from the Oxytetracyline Biosynthetic Pathway in Streptomyces rimosus Results in Novel Polyketides with Altered Chain Length
  作者：Nataša Perić-Concha、Branko Borovička、Paul F. Long、Daslav Hranueli、Peter G. Waterman、Iain S. Hunter

  DOI：10.1074/jbc.m503191200

  日期：2005.11

  Oxytetracycline (OTC) is a 19-carbon polyketide antibiotic made by Streptomyces rimosus. The otcC gene encodes an anhydrotetracycline oxygenase that catalyzes a hydroxylation of the anthracycline structure at position C-6 after biosynthesis of the polyketide backbone is completed. A recombinant strain of S. rimosus that was disrupted in the genomic copy of otcC synthesized a novel C-17 polyketide. This result indicates that the absence of the otcC gene product significantly influences the ability of the OTC "minimal" polyketide synthase to make a polyketide product of the correct chain length. A mutant copy of otcC was made by site-directed mutagenesis of three essential glycine codons located within the putative NADPH-binding domain. The mutant gene was expressed in Escherichia coli, and biochemical analysis confirmed that the gene product was catalytically inactive. When the mutant gene replaced the ablated gene in the chromosome of S. rimosus, the ability to make a 19-carbon backbone was restored, indicating that OtcC is an essential partner in the quaternary structure of the synthase complex.
• Uncovering the Enzymes that Catalyze the Final Steps in Oxytetracycline Biosynthesis
  作者：Peng Wang、Ghader Bashiri、Xue Gao、Michael R. Sawaya、Yi Tang

  DOI：10.1021/ja403516u

  日期：2013.5.15

  Tetracyclines are a group of natural products sharing a linearly fused four-ring scaffold, which is essential for their broad-spectrum antibiotic activities. Formation of the key precursor anhydrotetracycline 3 during oxytetracycline 1 biosynthesis has been previously characterized. However, the enzymatic steps that transform 3 into 1, including the additional hydroxylation at C5 and the final C5a-C11a reduction, have remained elusive. Here we report two redox enzymes, OxyS and OxyR, are sufficient to convert 3 to 1. OxyS catalyzes two sequential hydroxylations at C6 and C5 positions of 3 with opposite stereochemistry, while OxyR catalyzes the C5a-C11a reduction using F-420 as a cofactor to produce 1. The crystal structure of OxyS was obtained to provide insights into the tandem C6- and C5-hydroxylation steps. The substrate specificities of OxyS and OxyR were shown to influence the relative ratio of 1 and tetracycline 2.