2-Benzylsuccinate

• 分子结构分类: 有机化合物 - 苯丙烷和聚酮 - 苯丙酸
• 英文名称: 2-Benzylsuccinate
• 英文别名: 2-benzylbutanedioate
• 化学式: C11H10O4-2
• 分子量: 206.19
• InChiKey: GTOFKXZQQDSVFH-UHFFFAOYSA-L

计算性质

• 辛醇/水分配系数(LogP)：
  2.5
• 重原子数：
  15
• 可旋转键数：
  3
• 环数：
  1.0
• sp3杂化的碳原子比例：
  0.27
• 拓扑面积：
  80.3
• 氢给体数：
  0
• 氢受体数：
  4

反应信息

• 作为产物：
  描述：

  fumarate 、 甲苯 生成 2-Benzylsuccinate
  参考文献：
  名称：

  Anaerobic activation of toluene and o-xylene by addition to fumarate in denitrifying strain T

  摘要：

  使用菌株T进行厌氧实验，该菌株是一种可以将甲苯矿化为二氧化碳的脱氮细菌，实验结果表明，生长于甲苯的渗透细胞催化了甲苯与富马酸的加成反应，形成苯基琥珀酸。该反应不依赖于辅酶A（CoA）或ATP的存在。在CoA存在的情况下，还观察到苯基顺丁烯酸从苯基琥珀酸形成的反应。动力学研究表明，与硝酸全细胞悬浮液中的甲苯消耗比较，渗透细胞中甲苯和富马酸形成苯基琥珀酸的特定速率大于30％，这表明苯基琥珀酸的形成可能是菌株T厌氧降解甲苯的第一反应。使用氘标记的甲苯和气相色谱质谱技术表明，加成到富马酸过程中从甲苯甲基基团中提取的氢原子保留在苯基琥珀酸的琥珀酰基中。在本研究中，没有发现支持甲苯与乙酰辅酶A或琥珀酰辅酶A之前提出的反应的证据。菌株T生长于甲苯的渗透细胞还催化了将o-二甲苯加成到富马酸的反应，形成（2-甲基苯基）琥珀酸。o-二甲苯不是菌株T的生长底物，其转化可能是共代谢的。除了特定的反应速率，甲苯-富马酸加成反应的观察特性（即甲基氢原子的保留和不依赖于CoA和ATP）也适用于o-二甲苯-富马酸加成反应。因此，加成到富马酸可能是一种生化策略，用于厌氧激活一系列甲基苯。

  DOI：

  10.1128/jb.179.3.670-676.1997

文献信息

• Analysis of the Novel Benzylsuccinate Synthase Reaction for Anaerobic Toluene Activation Based on Structural Studies of the Product
  作者：Harry R. Beller、Alfred M. Spormann

  DOI：10.1128/jb.180.20.5454-5457.1998

  日期：1998.10.15

  Recent studies of anaerobic toluene catabolism have demonstrated a novel reaction for anaerobic hydrocarbon activation: the addition of the methyl carbon of toluene to fumarate to form benzylsuccinate. In vitro studies of the anaerobic benzylsuccinate synthase reaction indicate that the H atom abstracted from the toluene methyl group during addition to fumarate is retained in the succinyl moiety of benzylsuccinate. Based on structural studies of benzylsuccinate formed during anaerobic, in vitro assays with denitrifying, toluene-mineralizing strain T, we now report the following characteristics of the benzylsuccinate synthase reaction: (i) it is highly stereospecific, resulting in >95% formation of the (+)-benzylsuccinic acid enantiomer [( R )-2-benzyl-3-carboxypropionic acid], and (ii) active benzylsuccinate synthase does not contain an abstracted methyl H atom from toluene at the beginning or at the end of a catalytic cycle.

  摘要 最近对厌氧甲苯分解代谢的研究表明，厌氧碳氢化合物活化有一种新的反应：甲苯的甲基碳与富马酸加成形成琥珀酸苄酯。对厌氧琥珀酸苄酯合成酶反应的体外研究表明，在与富马酸加成过程中从甲苯甲基中抽取的 H 原子保留在琥珀酸苄酯的琥珀酰基中。根据对脱氮、甲苯矿化菌株 T 在厌氧、体外试验过程中形成的琥珀酸苄酯的结构研究，我们现在报告琥珀酸苄酯合成酶反应的以下特点：（i）它具有高度的立体特异性，导致 95% 的(+)-琥珀酸苄酯对映体形成[( R )-2-苄基-3-羧基丙酸]，以及(ii) 活性苄基琥珀酸合成酶在催化循环开始或结束时不包含从甲苯中抽取的甲基 H 原子。
• A Stable Organic Free Radical in Anaerobic Benzylsuccinate Synthase of Azoarcus sp. Strain T
  作者：Cynthia J. Krieger、Winfried Roseboom、Simon P.J. Albracht、Alfred M. Spormann

  DOI：10.1074/jbc.m009453200

  日期：2001.4

  The novel enzyme benzylsuccinate synthase initiates anaerobic toluene metabolism by catalyzing the addition of toluene to fumarate, forming benzylsuccinate. Based primarily on its sequence similarity to the glycyl radical enzymes, pyruvate formate-lyase and anaerobic ribonucleotide reductase, benzylsuccinate synthase was speculated to be a glycyl radical enzyme. In this report we use EPR spectroscopy to demonstrate for the first time that active benzylsuccinate synthase from the denitrifying bacterium Azoarcus sp. strain T harbors an oxygen-sensitive stable organic free radical. The EPR signal of the radical was centered at g = 2.0021 and was characterized by a major S-fold splitting of about 1.5 millitesla. The strong similarities between the EPR signal of the benzylsuccinate synthase radical and that of the glycyl radicals of pyruvate formate-lyase and anaerobic ribonucleotide reductase provide evidence that the benzylsuccinate synthase radical is located on a glycine residue, presumably glycine 828 in Azoarcus sp. strain T benzylsuccinate synthase.
• Biochemical and genetic characterization of benzylsuccinate synthase from<i>Thauera aromatica</i>: a new glycyl radical enzyme catalysing the first step in anaerobic toluene metabolism
  作者：Birgitta Leuthner、Christina Leutwein、Henk Schulz、Patric Hörth、Wolfgang Haehnel、Emile Schiltz、Hermann Schägger、Johann Heider

  DOI：10.1046/j.1365-2958.1998.00826.x

  日期：1998.4

  Toluene is anoxically degraded to CO2 by the denitrifying bacterium Thauera aromatica. The initial reaction in this pathway is the addition of fumarate to the methyl group of toluene, yielding benzylsuccinate as the first intermediate. We purified the enzyme catalysing this reaction, benzylsuccinate synthase (EC 4.1.99‐), and studied its properties. The enzyme was highly oxygen sensitive and contained a redox‐active flavin cofactor, but no iron centres. The native molecular mass was 220 kDa; four subunits of 94 (α), 90 (α′), 12 (β) and 10 kDa (γ) were detected on sodium dodecyl sulphate (SDS) gels. The N‐terminal sequences of the α‐ and α′‐subunits were identical, suggesting a C‐terminal degradation of half of the α‐subunits to give the α′‐subunit. The composition of native enzyme therefore appears to be α2β2γ2. A 5 kb segment of DNA containing the genes for the three subunits of benzylsuccinate synthase was cloned and sequenced. The masses of the predicted gene products correlated exactly with those of the subunits, as determined by electrospray mass spectrometry. Analysis of the derived amino acid sequences revealed that the large subunit of the enzyme shares homology to glycyl radical enzymes, particularly near the predicted radical site. The highest similarity was observed with pyruvate formate lyases and related proteins. The radical‐containing subunit of benzylsuccinate synthase is oxygenolytically cleaved at the site of the glycyl radical, producing the α′‐subunit. The predicted cleavage site was verified using electrospray mass spectrometry. In addition, a gene coding for an activating protein catalysing glycyl radical formation was found. The four genes for benzylsuccinate synthase and the activating enzyme are organized as a single operon; their transcription is induced by toluene. Synthesis of the predicted gene products was achieved in Escherichia coli in a T7‐promotor/polymerase system.
• Phototrophic utilization of toluene under anoxic conditions by a new strain of Blastochloris sulfoviridis
  作者：K. Zengler、Johann Heider、Ramon Rosselló-Mora、Friedrich Widdel

  DOI：10.1007/s002030050761

  日期：1999.9.27

  The capacity of anoxygenic phototrophic bacteria to utilize aromatic hydrocarbons was investigated in enrichment cultures with toluene. When mineral medium with toluene (provided in an inert carrier phase) was inoculated with activated sludge and incubated under infrared illumination (> 750 nm), a red-to-brownish culture developed. Agar dilution series indicated the dominance of two types of phototrophic bacteria. One type formed red colonies, had rod-shaped cells with budding division, and grew on benzoate but not on toluene. The other type formed yellow-to-brown colonies, had oval cells, and utilized toluene and benzoate. One strain of the latter type, ToP1, was studied in detail. Sequence analysis of the 16S rRNA gene and DNA-DNA hybridization indicated an affiliation of strain ToP1 with the species Blastochloris sulfoviridis, a member of the alpha-subclass of Proteobacteria. However. the type strain (DSM 729) of Blc. sulfoviridis grew neither on toluene nor on benzoate. Light-dependent consumption of toluene in the presence of carbon dioxide and formation of cell mass by strain ToP1 were demonstrated in quantitative growth experiments. Strain ToP1 is the first phototrophic bacterium shown to utilize an aromatic hydrocarbon. In the supernatant of toluene-grown cultures and in cell-free extracts incubated with toluene and fumarate, the formation of benzylsuccinate was detected. These findings indicate that the phototrophic bacterium activates toluene anaerobically by the same mechanism that has been reported for denitrifying and sulfate-reducing bacteria. The natural abundance of phototrophic bacteria with the capacity for toluene utilization was examined in freshwater habitats. Counting series revealed that up to around 1% (1.8 x 10(5) cells per gram dry mass of sample) of the photoheterotrophic population cultivable with acetate grew on toluene.
• Deuterium Isotope Effects in the Unusual Addition of Toluene to Fumarate Catalyzed by Benzylsuccinate Synthase
  作者：Lei Li、E. Neil G. Marsh

  DOI：10.1021/bi061117o

  日期：2006.11.1

  The first step in the anaerobic metabolism of toluene is a highly unusual reaction: the addition of toluene across the double bond of fumarate to produce (R)- benzylsuccinate, which is catalyzed by benzylsuccinate synthase. Benzylsuccinate synthase is a member of the glycyl radical-containing family of enzymes, and the reaction is initiated by abstraction of a hydrogen atom from the methyl group of toluene. To gain insight into the free energy profile of this reaction, we have measured the kinetic isotope effects on V-max and V-max/K-m when deuterated toluene is the substrate. At 30 degrees C the isotope effects are 1.7 +/- 0.2 and 2.9 +/- 0.1 on V-max and V-max/K-m, respectively; at 4 degrees C they increase slightly to 2.2 +/- 0.2 and 3.1 +/- 0.1, respectively. We compare these results with the theoretical isotope effects on V-max and V-max/K-m that are predicted from the free energy profile for the uncatalyzed reaction, which has previously been computed using density functional theory [Himo, F. (2002) J. Phys. Chem. B 106, 7688- 7692]. The comparison allows us to draw some conclusions on how the enzyme may catalyze this unusual reaction.