(3S)-3-hydroxy-2-oxobutanoic acid

• 分子结构分类: 有机化合物 - 有机酸及其衍生物 - 酮酸及其衍生物
• 英文名称: (3S)-3-hydroxy-2-oxobutanoic acid
• 化学式: C₄H₆O₄
• 分子量: 118.089
• InChiKey: QWZIITCYKKSZGN-REOHCLBHSA-N

计算性质

• 辛醇/水分配系数(LogP)：
  -0.6
• 重原子数：
  8
• 可旋转键数：
  2
• 环数：
  0.0
• sp3杂化的碳原子比例：
  0.5
• 拓扑面积：
  74.6
• 氢给体数：
  2
• 氢受体数：
  4

反应信息

• 作为产物：
  描述：

  D-苏氨酸 在 2,2'-(1,2-肼二亚基)二[3-乙基-2,3-二氢-6-苯并噻唑磺酸 、 recombinant Trigonopsis variabilis D-amino acid oxidase 、 horseradish peroxidase 作用下， 生成 (3S)-3-hydroxy-2-oxobutanoic acid
  参考文献：
  名称：

  The role of residues Arg169 and Arg220 in intersubunit interactions of yeast D-amino acid oxidase

  摘要：

  来自变叶酵母（Trigonopsis variabilis）的 D-氨基酸氧化酶（EC 1.4.3.3，TvDAAO）是由两个相同的亚基组成的二聚体。该酶的二聚体结构由 12 个（6 对）氢键稳定，每个亚基的 Arg169 和 Arg220 残基参与了 8 个氢键。我们制备了 TvDAAO 的 Arg169Glu 和 Arg(169,220)Ala 突变体。这两种突变体酶在大肠杆菌细胞中表达为不溶解但具有催化活性的包涵体。在亚基间界面引入氨基酸取代导致底物特异性发生变化，热稳定性大大降低。

  DOI：

  10.1007/s11172-010-0072-9

文献信息

• Mutant d-amino acid oxidase with higher catalytic efficiency toward d-amino acids with bulky side chains
  作者：N. V. Komarova、I. V. Golubev、S. V. Khoronenkova、V. I. Tishkov

  DOI：10.1007/s11172-012-0193-4

  日期：2012.7

  d-Amino acid oxidase from the yeast Trigonopsis variabilis (TvDAAO) is widely used in fine organic synthesis, including the preparation of unnatural l-amino acids and α-keto acids. The analysis of the three-dimensional structure of TvDAAO was carried out with the aim of producing the enzyme specific to d-amino acids with bulky side chains. The analysis revealed the residue Phe54 at the entrance to the active site, which controls the substrate access to this site. The residue Phe54 was replaced by residues Ala, Ser, and Tyr. The cultivation of recombinant E. coli strains expressing TvDAAO mutants showed that the mutein with the Phe54Ala substitution had very low stability. Thus, the inactivation of the enzyme occured within 10 min after the cell disruption. The Phe54Ser TvDAAO and Phe54Tyr TvDAAO mutants were obtained as homogeneous preparations, and their thermal stability and catalytic properties were investigated. The introduction of Phe54Ser and Phe54Tyr substitutions resulted in additional stabilization of the protein macromolecule compared to the wild-type TvDAAO. Thus, the half-inactivation time for the mutant enzymes at 54 °C increased by a factor of 1.5 and 2, respectively. As in the case of wild-type TvDAAO, the thermal inactivation of the muteins proceeds via a two-step dissociative mechanism. The introduction of mutations led to a strong change in the substrate specificity profile. The mutants have no activity toward a series of d-amino acids (Phe54Ser TvDAAO toward d-Ala, d-Ser, d-Val, and d-Thr; Phe54Tyr TvDAAO toward d-Ser, d-Tyr, d-Thr, and d-Lys). The catalytic efficiency (the k cat/K M ratio) of the Phe54Ser TvDAAO mutant toward d-amino acids with bulky side chains (d-Lys, d-Asn, d-Phe, d-Tyr, d-Trp, and d-Leu) increased from 2.4 to 7.3 times.

  来自变叶酵母（Trigonopsis variabilis）的 d-氨基酸氧化酶（TvDAAO）被广泛用于精细有机合成，包括制备非天然 l-氨基酸和 α-酮酸。对 TvDAAO 的三维结构进行了分析，目的是生产出对具有粗大侧链的 d- 氨基酸具有特异性的酶。分析结果显示，活性位点入口处的残基 Phe54 控制着底物进入该位点。Phe54残基被Ala、Ser和Tyr残基取代。表达 TvDAAO 突变体的重组大肠杆菌菌株的培养结果表明，Phe54Ala 取代后的静音蛋白稳定性非常低。因此，该酶在细胞破坏后 10 分钟内就失活了。研究人员获得了 Phe54Ser TvDAAO 和 Phe54Tyr TvDAAO 突变体的均质制备方法，并对其热稳定性和催化特性进行了研究。与野生型 TvDAAO 相比，Phe54Ser 和 Phe54Tyr 的引入使蛋白质大分子更加稳定。因此，突变体酶在 54 °C 的半失活时间分别增加了 1.5 倍和 2 倍。与野生型 TvDAAO 的情况一样，静音素的热失活也是通过两步解离机制进行的。突变的引入导致底物特异性发生了很大变化。突变体对一系列 d-氨基酸没有活性（Phe54Ser TvDAAO 对 d-Ala、d-Ser、d-Val 和 d-Thr；Phe54Tyr TvDAAO 对 d-Ser、d-Tyr、d-Thr 和 d-Lys）。Phe54Ser TvDAAO 突变体对具有笨重侧链的 d-氨基酸（d-Lys、d-Asn、d-Phe、d-Tyr、d-Trp 和 d-Leu）的催化效率（k cat/K M 比率）从 2.4 倍提高到 7.3 倍。
• Characterization of d-amino acid aminotransferase from Lactobacillus salivarius
  作者：Jyumpei Kobayashi、Yasuhiro Shimizu、Yuta Mutaguchi、Katsumi Doi、Toshihisa Ohshima

  DOI：10.1016/j.molcatb.2013.04.013

  日期：2013.10

  We searched a UniProt database of lactic acid bacteria in an effort to identify D-amino acid metabolizing enzymes other than alanine racemase. We found a D-amino acid aminotransferase (D-AAT) homologous gene (UniProt ID: Q1WRM6) in the genome of Lactobacillus salivarius. The gene was then expressed in Escherichia coli, and its product exhibited transaminase activity between D-alanine and alpha-ketoglutarate. This is the first characterization of a D-AAT from a lactic acid bacterium. L. salivarius D-AAT is a homodimer that uses pyridoxal-5'-phosphate (PLP) as a cofactor; it contains 0.91 molecules of PLP per subunit. Maximum activity was seen at a temperature of 60 degrees C and a pH of 6.0. However, the enzyme lost no activity when incubated for 30 min at 30 degrees C and pH 5.5 to 9.5, and retained half its activity when incubated at pH 4.5 or 11.0 under the same conditions. Double reciprocal plots of the initial velocity and D-alanine concentrations in the presence of several fixed concentrations of alpha-ketoglutarate gave a series of parallel lines, which is consistent with a Ping-Pong mechanism. The K-m values for D-alanine and alpha-ketoglutarate were 1.05 and 3.78 mM, respectively. With this enzyme, D-allo-isoleucine exhibited greater relative activity than D-alanine as the amino donor, while alpha-ketobutylate, glyoxylate and indole-3-pyruvate were all more preferable amino acceptors than alpha-ketoglutarate. The substrate specificity of L. salivarius D-AAT thus differs greatly from those of the other D-AATs so far reported. (c) 2013 Elsevier B.V. All rights reserved.
• Role of the active site residues arginine-216 and arginine-237 in the substrate specificity of mammalian d-aspartate oxidase
  作者：Masumi Katane、Yasuaki Saitoh、Kazuhiro Maeda、Toshihiko Hanai、Masae Sekine、Takemitsu Furuchi、Hiroshi Homma

  DOI：10.1007/s00726-010-0658-4

  日期：2011.2

  d-Aspartate oxidase (DDO) and d-amino acid oxidase (DAO) are flavin adenine dinucleotide-containing flavoproteins that catalyze the oxidative deamination of d-amino acids. Unlike DAO, which acts on several neutral and basic d-amino acids, DDO is highly specific for acidic d-amino acids. Based on molecular modeling and simulated annealing docking analyses, a recombinant mouse DDO carrying two substitutions (Arg-216 to Leu and Arg-237 to Tyr) was generated (R216L-R237Y variant). This variant and two previously constructed single-point mutants of mouse DDO (R216L and R237Y variants) were characterized to investigate the role of Arg-216 and Arg-237 in the substrate specificity of mouse DDO. The R216L-R237Y and R216L variants acquired a broad specificity for several neutral and basic d-amino acids, and showed a considerable decrease in activity against acidic d-amino acids. The R237Y variant, however, did not show any additional specificity for neutral or basic d-amino acids and its activity against acidic d-amino acids was greatly reduced. The kinetic properties of these variants indicated that the Arg-216 residue is important for the catalytic activity and substrate specificity of mouse DDO. However, Arg-237 is, apparently, only marginally involved in substrate recognition, but is important for catalytic activity. Notably, the substrate specificity of the R216L-R237Y variant differed significantly from that of the R216L variant, suggesting that Arg-237 has subsidiary effects on substrate specificity. Additional experiments using several DDO and DAO inhibitors also suggested the involvement of Arg-216 in the substrate specificity and catalytic activity of mouse DDO and that Arg-237 is possibly involved in substrate recognition by this enzyme. Collectively, these results indicate that Arg-216 and Arg-237 play crucial and subsidiary role(s), respectively, in the substrate specificity of mouse DDO.
• The role of residues Arg169 and Arg220 in intersubunit interactions of yeast D-amino acid oxidase
  作者：N. V. Cherskova、S. V. Khoronenkova、V. I. Tishkov

  DOI：10.1007/s11172-010-0072-9

  日期：2010.1

  D-Amino acid oxidase from the yeast Trigonopsis variabilis (EC 1.4.3.3, TvDAAO) exists as a dimer consisting of two identical subunits. The dimeric structure of the enzyme is stabilized by 12 (six pairs) hydrogen bonds, the residues Arg169 and Arg220 of each subunit being involved in eight hydrogen bonds. The Arg169Glu and Arg(169,220)Ala mutants of TvDAAO were prepared. Both mutant enzymes were expressed in E. coli cells as insoluble but catalytically active inclusion bodies. The introduction of amino acid substitutions at the intersubunit interface resulted in a change in the substrate specificity profile and a strong decrease in thermal stability.

  来自变叶酵母（Trigonopsis variabilis）的 D-氨基酸氧化酶（EC 1.4.3.3，TvDAAO）是由两个相同的亚基组成的二聚体。该酶的二聚体结构由 12 个（6 对）氢键稳定，每个亚基的 Arg169 和 Arg220 残基参与了 8 个氢键。我们制备了 TvDAAO 的 Arg169Glu 和 Arg(169,220)Ala 突变体。这两种突变体酶在大肠杆菌细胞中表达为不溶解但具有催化活性的包涵体。在亚基间界面引入氨基酸取代导致底物特异性发生变化，热稳定性大大降低。