Non‐Heme Hydroxylase Engineering For Simple Enzymatic Synthesis of L‐threo‐Hydroxyaspartic Acid
Non‐Heme Hydroxylase Engineering For Simple Enzymatic Synthesis of L‐threo‐Hydroxyaspartic Acid
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用于简单酶法合成 LâthreoâHydroxyaspartic Acid 的非血红素羟化酶工程
DOI：
10.1002/cbic.200700557
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发表时间：
2008
期刊：
影响因子：
3.2
通讯作者：
Marahiel
中科院分区：
文献类型：
--
作者：
Strieker;Marahiel
L-苏型-羟基天冬氨酸（L-THA，1）是L-天冬氨酸的β-羟基化形式（2），目前具有药用价值，因为它抑制L-天冬酰胺合成酶，[1]是凝血级联中几种蛋白质的关键成分，[2]并抑制兴奋性氨基酸（EAA）转运蛋白作为L-谷氨酸模拟物的功能。[3]这些功能中的最后一个更重要，因为L-谷氨酸作为哺乳动物中枢神经系统（CNS）中的主要神经递质发挥关键作用，并通过激活广泛种类的EAA受体参与多样和复杂的神经元通信。[4]由于其过度激活这些受体的潜力，L-谷氨酸可导致急性损伤或慢性疾病中的CNS损伤。[5]因此，由EAA转运蛋白进行的细胞外L-谷氨酸浓度的调节至关重要。一个现成的来源1可以帮助进一步调查这些转运蛋白和L-谷氨酸介导的信号转导过程的复杂性。先前已经描述了赤型-和苏型-羟基天冬氨酸的复合非对映异构体混合物的各种合成路线。[4]这些制备是迂回和昂贵的，使得更有效的合成1所需的。酶催化提供了一种替代方法，但据我们所知，尚未描述直接催化2至1的β-羟基化的羟化酶。因此，我们应用合理的蛋白质设计方法来完成这项任务。基于3D结构信息的蛋白质工程已经成为生物催化酶操作的公认工具，[7]我们使用这种方法将天冬酰胺加氧酶（AsnO）的底物特异性从L-Asn改变为L-Asp。AsnO参与天蓝色链霉菌中钙依赖性抗生素（CDA）的生物合成[8]，是一种Fe 2+和α-酮戊二酸依赖性（α KG依赖性）羟化酶，专门催化L-苏型-羟基天冬酰胺（3）的合成，用作CDA的结构单元。[9]AsnO因此提供具有所需立体化学的氨基酸，但不接受2作为底物。在以前的研究中，[9]解决了与3和琥珀酸盐复合的AsnO的晶体结构（PDB ID：2 OG 7），并鉴定了底物结合残基（方案1）。残基Asp 241的侧链与L-Asn底物的羧酰胺基团的NH 2结合。我们假设，该残基的定点突变为天冬酰胺将产生天冬氨酸侧链的结合位点。Asp 241的突变不影响α-羧基和α-氨基配位残基（Glu 125，Asn 146，Arg 305）的立体化学结构，因此可以保留其立体化学结构。[9]变体AsnO D241 N表达为His 7标记的融合物，并纯化为可溶性蛋白，产量为5-6 mg/L细菌培养物。Ni-NTA亲和层析后，SDS-PAGE分析表明纯度> 95%（参见支持信息中的图S1）。为了在分析规模上评价其活性，将纯化的酶与2、（NH 4）2FeACHTUNGTRENNUNG（SO 4）2（作为亚铁辅因子的来源）和αKG共底物在不同温度下孵育。监测反应，并通过HPLC-MS分析通过扫描2（[M+ H]+= 134.05 Da）及其羟基化形式1（[M+ H]+= 150.04 Da）的质量来验证反应产物的身份。2与AsnO D241 N孵育后的HPLC-MS色谱图（图S2）显示...
L-threo-Hydroxyaspartic acid (L-THA, 1), the β-hydroxylated form of L-aspartic acid (2), is of current medicinal interest because it inhibits L-asparagine synthetase,[1] is a key constituent of several proteins in the blood-clotting cascade,[2] and inhibits the function of excitatory amino acid (EAA) transporters as an L-glutamic acid mimic.[3] The last of these functions is of greater importance, as L-glutamate plays a key role as a primary neurotransmitter in the mammalian central nervous system (CNS) and participates in diverse and complex neuronal communication by activating a broad assortment of the EAA receptors.[4] With its potential to overactivate these receptors, L-glutamate can contribute to CNS damage in acute injuries or chronic diseases.[5] Thus, regulation of extracellular L-glutamate concentration, carried out by the EAA transporters, is crucial. A readily available source of 1 could help in further investigations of these transporters and of the complexity of L-glutamate-mediated signaling processes. Various synthetic routes to complex diastereomeric mixtures of erythro-and threo-hydroxyaspartic acid have been described previously.[4] These preparations are circuitous and expensive, making a more efficient synthesis of 1 desirable. Enzymatic catalysis provides an alternative approach, but, to the best of our knowledge, no hydroxylase that directly catalyzes the β-hydroxylation of 2 to 1 has been described. We therefore applied a rational protein design approach to fulfill this task. Protein engineering, based on 3D-structural information, has become an accepted tool for the manipulation of enzymes for biocatalysis,[7] and we used this method to alter the substrate specificity of an asparagine oxygenase (AsnO) from L-Asn to L-Asp. AsnO, involved in the biosynthesis of calcium-dependent antibiotics (CDAs) in Streptomyces coelicolor,[8] is an Fe2+-and α-ketoglutarate-dependent (αKG-dependent) hydroxylase, which exclusively catalyzes the synthesis of L-threo-hydroxyasparagine (3), which is used as a CDA building block.[9] AsnO therefore provides an amino acid with the desired stereochemistry, but does not accept 2 as a substrate. In previous studies,[9] the crystal structure (PDB ID: 2OG7) of AsnO in complexation with 3 and succinate was solved, and the substrate binding residues were identified (Scheme 1). The side chain of residue Asp241 binds to the NH2 of the carboxamide group of the L-Asn substrate. We hypothesized that site-directed mutagenesis of this residue to asparagine would yield a binding site for an aspartate side chain. Retention of stereochemistry would also be expected, as the α-carboxy and α-amino coordinating residues (Glu125, Asn146, Arg305) are unaffected by the mutagenesis of Asp241.The site-directed mutagenesis of residue Asp241 to Asn was conducted by use of the asnO-containing expression vector as the template.[9] The variant—AsnO D241N—was expressed as a His7-tagged fusion and purified as soluble protein, with yields of 5–6 mg per liter of bacterial culture. After Ni-NTA affinity chromatography, SDS-PAGE analysis indicated> 95% purity (see Figure S1 in the Supporting Information). To evaluate its activity on an analytical scale, purified enzyme was incubated with 2,(NH4) 2FeACHTUNGTRENNUNG (SO4) 2 (as source of the ferrous iron cofactor), and αKG cosubstrate at different temperatures. The reaction was monitored, and the identity of the reaction product was verified by HPLC-MS analysis by scanning for the masses of 2 ([M+ H]+= 134.05 Da) and its hydroxylated form 1 ([M+ H]+= 150.04 Da). The HPLC-MS chromatogram (FigureS2) of the assay after incubation of 2 with AsnO D241N revealed …
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L-天冬酰胺生物合成的潜在抑制剂。
影响因子：
7.3
作者：
M. Mokotoff;J. F. Bagaglio;B. Parikh
通讯作者：
B. Parikh
酰基转移酶 II 酶解 β-甲基天冬氨酸
DOI：
--
发表时间：
1959
期刊：
影响因子：
--
作者：
L. Benoiton;S. M. Birnbaum;M. Winitz;J. Greenstein
通讯作者：
J. Greenstein
作为谷氨酸转运蛋白阻断剂的光学纯 β-羟基天冬氨酸衍生物的合成。
影响因子：
2.7
作者：
K. Shimamoto;Y. Shigeri;Y. Yasuda‐Kamatani;B. Lebrun;N. Yumoto;T. Nakajima
通讯作者：
T. Nakajima
影响因子：
4
作者：
Olney, JW
通讯作者：
Olney, JW
影响因子：
3.6
作者：
Hafez, AM;Dudding, T;Lectka, T
通讯作者：
Lectka, T