N-acetyl-L-aspartate

• 分子结构分类: 有机化合物 - 有机酸及其衍生物 - 羧酸及其衍生物
• 英文名称: N-acetyl-L-aspartate
• 英文别名: N-acetyl aspartate；N-acetylaspartate；NAA；(2S)-2-acetamidobutanedioate
• 化学式: C₆H₇NO₅
• 分子量: 173.125
• InChiKey: OTCCIMWXFLJLIA-BYPYZUCNSA-L

计算性质

• 辛醇/水分配系数(LogP)：
  0.2
• 重原子数：
  12
• 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：
  109
• 氢给体数：
  1
• 氢受体数：
  5

反应信息

• 作为反应物：
  描述：

  L-谷氨酸 、 N-acetyl-L-aspartate 在 乙二醇双(2-氨基乙基醚)四乙酸 、 His-tagged RIMKLA 、 L-[U-⁽¹⁴⁾C]glutamate 、 MgATP 、 magnesium chloride 、 Cleland's reagent 、 bovine serum albumin 作用下， 反应 0.5h， 生成 N-acetylaspartylglutamate 、 二磷酸腺苷
  参考文献：
  名称：

  Molecular Identification of N-Acetylaspartylglutamate Synthase and β-Citrylglutamate Synthase

  摘要：

  The purpose of the present work was to determine the identity of the enzymes that synthesize N-acetylaspartylglutamate (NAAG), the most abundant dipeptide present in vertebrate central nervous system (CNS), and beta-citrylglutamate, a structural analogue of NAAG present in testis and immature brain. Previous evidence suggests that NAAG is not synthesized on ribosomes but presumably is synthesized by a ligase. As attempts to detect this ligase in brain extracts failed, we searched the mammalian genomes for putative enzymes that could catalyze this type of reaction. Mammalian genomes were found to encode two putative ligases homologous to Escherichia coli RIMK, which ligates glutamates to the C terminus of ribosomal protein S6. One of them, named RIMKLA, is almost exclusively expressed in the CNS, whereas RIMKLB, which shares 65% sequence identity with RIMKLA, is expressed in CNS and testis. Both proteins were expressed in bacteria or HEK293T cells and purified. RIMKLA catalyzed the ATP-dependent synthesis of N-acetylaspartylglutamate from N-acetylaspartate and L-glutamate. RIMKLB catalyzed this reaction as well as the synthesis of beta-citrylglutamate. The nature of the reaction products was confirmed by mass spectrometry and NMR. RIMKLA was shown to produce stoichiometric amounts of NAAG and ADP, in agreement with its belonging to the ATP-grasp family of ligases. The molecular identification of these two enzymes will facilitate progress in the understanding of the function of NAAG and beta-citrylglutamate.

  DOI：

  10.1074/jbc.m110.152629
• 作为产物：
  描述：

  (2R)-N-[3-(2-acetylsulfanylethylimino)-3-oxidopropyl]-4-[[[(2R,3S,4R,5R)-5-(6-aminopurin-9-yl)-4-hydroxy-3-phosphonatooxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-hydroxyphosphoryl]oxy-2-hydroxy-3,3-dimethylbutanimidate 、 aspartate anion 生成 coenzyme A 、 氢(+1)阳离子 、 N-acetyl-L-aspartate
  参考文献：
  名称：

  Molecular identification of aspartate N-acetyltransferase and its mutation in hypoacetylaspartia

  摘要：

  大脑特异性化合物 NAA（N-乙酰天冬氨酸）几乎只存在于神经元中，其浓度约为 20 mM。通过 MRS（磁共振波谱）测定患者体内 NAA 的含量，以评估神经元的密度和健康状况。催化 NAA 合成的 NAT（N-乙酰转移酶）的分子特性一直不为人知，因为这种酶是膜结合的，难以纯化。数据库搜索表明，在人类和小鼠基因组编码的推定 NAT（即与已知 NAT 同源但催化活性未定性的蛋白质）中，有两种几乎只在大脑中表达，即 NAT8L 和 NAT14。在 HEK-293T [表达 SV40（猿猴病毒 40）大 T 抗原的人类胚胎肾脏-293 细胞]中进行的转染研究表明，NAT8L 可催化 L-天门冬氨酸和乙酰-CoA合成 NAA，而 NAT14 则不行。NAT8L 的特异性、对天冬氨酸的 Km 值及其对去垢剂的敏感性与脑 Asp-NAT 的描述相似。对表达重组 NAT8L 的 CHO（中国仓鼠卵巢）细胞和神经元进行的共聚焦显微镜分析表明，NAT8L 与 ER（内质网）相关，但与线粒体无关。对已知唯一缺乏 NAA 的患者的 NAT8L 基因进行突变搜索后发现，该基因存在一个 19 bp 的同源缺失，导致阅读框发生变化，无法产生功能蛋白。我们的结论是，NAT8L是一种神经元特异性蛋白，负责NAA的合成，在原发性NAA缺乏症（低乙酰天冬氨酸症）中发生突变。这种酶的分子鉴定将为阐明神经元中这种最丰富的氨基酸衍生物的功能以及诊断其他患者的低乙酰天冬氨酸症提供新的视角。

  DOI：

  10.1042/bj20091024

文献信息

• New T530C mutation in the aspartoacylase gene caused Canavan disease with no correlation between severity and N-acetylaspartate excretion
  作者：Valentina Di Pietro、Ugo Cavallari、Angela M. Amorini、Giacomo Lazzarino、Salvatore Longo、Carlo Poggiani、Pietro Cavalli、Barbara Tavazzi

  DOI：10.1016/j.clinbiochem.2013.09.004

  日期：2013.12

  ASPA gene in both sibs. A total loss of enzymatic activity was also recorded. CONCLUSIONS The substitution T530C (Ile177Thr) results in a novel missense mutation causing a CD phenotype with severe clinical characteristics. This mutation was not previously described in the literature. In these two sibs, urinary concentration of NAA appears to correlate inversely to symptom severity and CD progression

  目的Canavan病（OMIM 271900）是一种严重的常染色体隐性神经退行性疾病，其特征是大脑海绵状变性，并由天冬氨酸酰化酶（ASPA）编码基因的突变引起。该酶负责脑特异性化合物N-乙酰天门冬氨酸（NAA）的催化。设计与方法我们报道了两名埃及兄弟姐妹患者，他们被怀疑患有Canavan病（CD），临床表现恶化，白质变性，巨脑和严重智力障碍。患者接受了磁共振成像（MRI）和生物流体样品（血清和尿液）中NAA的生化分析。随后，为了确定引起这两个同胞中CD的突变，进行了分子生物学检查。结果MRI的发现和高NAA排泄（分别为4个月和4岁患者尿液中的1378.5和680.1μmolNAA/ mmolcreatinine的定量）证实了CD的诊断并促使寻找相关突变。分子生物学分析显示，两个同胞中ASPA基因第4外显子的取代T530C（Ile177Thr）是纯合的。还记录了酶活性的完全丧失。结论替代T5
• Identification of the zinc binding ligands and the catalytic residue in human aspartoacylase, an enzyme involved in Canavan disease
  作者：S. Herga、J.-G. Berrin、J. Perrier、A. Puigserver、T. Giardina

  DOI：10.1016/j.febslet.2006.09.056

  日期：2006.10.30

  Sequence alignment showed that this enzyme belongs to the carboxypeptidase metalloprotein family having the conserved motif H21xxE24(91aa)H116. We further investigated the active site of hASP by performing modelling studies and site‐directed mutagenesis. His21, Glu24 and His116 were identified here for the first time as the residues involved in the zinc‐binding process. In addition, mutations involving the

  Canavan 病是一种常染色体隐性神经退行性疾病，由缺乏天冬氨酸酰化酶引起，天冬氨酸酰化酶可将 N-乙酰天冬氨酸 (NAA) 降解为醋酸盐和天冬氨酸。为了研究人类天冬氨酸酰化酶 (hASP) 的作用机制，该酶在异源大肠杆菌系统中表达并表征。发现重组蛋白的分子量为 36 kDa，动力学常数 K m 和 k cat 分别为 0.20 ± 0.03 mM 和 14.22 ± 0.48 s-1。序列比对表明该酶属于具有保守基序 H21xxE24(91aa)H116 的羧肽酶金属蛋白家族。我们通过进行建模研究和定点诱变进一步研究了 hASP 的活性位点。他的21，Glu24 和 His116 在这里首次被鉴定为参与锌结合过程的残基。此外，涉及 Glu178Gln 和 Glu178Asp 残基的突变导致酶活性丧失。野生型和 Glu178Asp 具有相同的 K m 但不同的 k cat 值的发现证实了
• Clinically Distinct Phenotypes of Canavan Disease Correlate with Residual Aspartoacylase Enzyme Activity
  作者：Marisa I Mendes、Desirée EC Smith、Ana Pop、Pascal Lennertz、Matilde R Fernandez Ojeda、Warsha A Kanhai、Silvy JM van Dooren、Yair Anikster、Ivo Barić、Caroline Boelen、Jaime Campistol、Lonneke de Boer、Ariana Kariminejad、Hulya Kayserili、Agathe Roubertie、Krijn T Verbruggen、Christine Vianey-Saban、Monique Williams、Gajja S Salomons

  DOI：10.1002/humu.23181

  日期：2017.5

  measuring the released aspartic acid by LC-MS/MS. Clinical data were obtained for 11 patients by means of questionnaires. Four patients presented with a non-typical clinical picture or with the milder form of CD, whereas seven presented with severe CD. The mutations found in the mild patients corresponded to the variants with the highest residual enzyme activities, suggesting that this assay can help evaluate

  我们描述了14位ASPA中12个新的错义突变的患者，ASPA是导致Canavan疾病（CD）的基因。我们开发了一种方法来研究这12个变体对天冬氨酸酰化酶功能的影响-N-乙酰基-1-天冬氨酸（NAA）水解为天冬氨酸和乙酸盐。将野生型ASPA开放阅读框（ORF）和包含每个变体的ORFs转染到HEK293细胞中。通过将细胞裂解物与NAA孵育并通过LC-MS / MS测量释放的天冬氨酸来测定酶活性。通过问卷调查获得了11例患者的临床数据。4例患者表现出非典型的临床表现或较轻度的CD，而7例表现为严重CD。在轻度患者中发现的突变对应于具有最高残留酶活性的变体，提示这种检测方法可以帮助评估在非典型表现患者中发现的未知变体。我们已经检测到临床表现，酶活性和CD基因型之间的相关性。
• Defective N-acetylaspartate catabolism reduces brain acetate levels and myelin lipid synthesis in Canavan's disease
  作者：Chikkathur N. Madhavarao、Peethambaran Arun、John R. Moffett、Sylvia Szucs、Sankar Surendran、Reuben Matalon、James Garbern、Diana Hristova、Anne Johnson、Wei Jiang、M. A. Aryan Namboodiri

  DOI：10.1073/pnas.0409184102

  日期：2005.4.5

  Canavan's disease (CD) is a fatal, hereditary disorder of CNS development that has been linked to mutations in the gene for the enzyme aspartoacylase (ASPA) (EC 3.5.1.15). ASPA acts to hydrolyze N -acetylaspartate (NAA) into l -aspartate and acetate, but the connection between ASPA deficiency and the failure of proper CNS development is unclear. We hypothesize that one function of ASPA is to provide acetate for the increased lipid synthesis that occurs during postnatal CNS myelination. The gene encoding ASPA has been inactivated in the mouse model of CD, and here we show significant decreases in the synthesis of six classes of myelinassociated lipids, as well as reduced acetate levels, in the brains of these mice at the time of peak postnatal CNS myelination. Analysis of the lipid content of white matter from a human CD patient showed decreased cerebroside and sulfatide relative to normal white matter. These results demonstrate that myelin lipid synthesis is significantly compromised in CD and provide direct evidence that defective myelin synthesis, resulting from a deficiency of NAA-derived acetate, is involved in the pathogenesis of CD.

  Canavan病（CD）是一种致命的遗传性中枢神经系统发育障碍，已与编码酶天冬氨酸乙酰化酶（ASPA）（EC 3.5.1.15）的基因突变相关联。ASPA的作用是将N-乙酰天冬氨酸（NAA）水解为L-天冬氨酸和乙酸，但ASPA缺乏与中枢神经系统发育不良的关系尚不清楚。我们假设ASPA的一个功能是为出生后中枢神经系统髓鞘化过程中发生的脂质合成提供乙酸。在CD的小鼠模型中失活了编码ASPA的基因，并且我们在这里展示这些小鼠的大脑在出生后中枢神经系统髓鞘化的高峰期时，六种类别的髓鞘相关脂质合成显著降低，以及乙酸水平降低。对CD患者白质的脂质含量分析显示，相对于正常白质，鞘脂和硫酸鞘脂降低。这些结果表明，在CD中髓鞘脂质合成显著受损，并且提供了直接证据表明，由于NAA衍生的乙酸缺乏导致的髓鞘合成缺陷参与了CD的发病机制。
• Characterization of the N-acetylaspartate biosynthetic enzyme from rat brain
  作者：C. N. Madhavarao、C. Chinopoulos、K. Chandrasekaran、M. A. A. Namboodiri

  DOI：10.1046/j.1471-4159.2003.01905.x

  日期：——

  the pellet of the rat brain homogenate (26 000 g for 1 h) for the recovery of maximum activity and partially purified using three protein separation methods: DEAE anion exchange chromatography, continuous elution native gel electrophoresis and size-exclusion high performance liquid chromatography. Asp-NAT activity and the optical density pattern of the eluted protein from size-exclusion column indicated

  天冬氨酸N-乙酰基转移酶（Asp-NAT； EC 2.3.1.17）的活性在通过Percoll梯度离心法制备的高度纯化的完整线粒体以及在通过蔗糖密度梯度离心法分离的Percoll纯化的线粒体中获得的三个亚组分中均发现。柠檬酸合酶用作线粒体的标记酶。在线粒体和突触体组分中，Asp-NAT和柠檬酸合酶可恢复活性的比例相当，但在含有髓磷脂的组分中却没有。从大鼠脑匀浆的沉淀物中溶解Asp-NAT（26 000 g，持续1 h）以恢复最大活性，并使用三种蛋白质分离方法部分纯化：DEAE阴离子交换色谱法，连续洗脱天然凝胶电泳和尺寸排阻高效液相色谱。从大小排阻柱洗脱的蛋白的Asp-NAT活性和光密度图谱表明单个大蛋白（约670 kDa），在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上显示至少10条带，表明有酶复合物。这种看似多亚基的复合物Asp-NAT对离子扰动稳定，但易受疏水性扰动的影响。10 mm 3-[（（3-胆酰胺基丙基）二甲基氨]