• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Journal of Molecular Biology >Evidence that human histidine triad nucleotide binding protein 3 (Hint3) is a distinct branch of the histidine triad (HIT) superfamily
【24h】

Evidence that human histidine triad nucleotide binding protein 3 (Hint3) is a distinct branch of the histidine triad (HIT) superfamily

机译：人类组氨酸三联体核苷酸结合蛋白3（Hint3）是组氨酸三联体（HIT）超家族的明显分支的证据

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Human Hint3 (hHint3) has been classified as a member of the histidine triad nucleotide (Hint) binding protein subfamily. While Hint1 is ubiquitously expressed by both eukaryotes and prokaryotes, Hint3 is found only in eukaryotes. Previously, our laboratory has characterized and compared the aminoacyl-adenylate and nucleoside phosphoramidate hydrolase activity of hHint1 and Escherichia coli hinT. In this study, hHint3-1(Ala36) and its single nucleotide polymorphism, hHint3-2 (A36G variant), were cloned, overexpressed, and purified. Steady-state kinetic studies with a synthetic fluorogenic indolepropinoic acyl-adenylate (AIPA) and with a series of fluorogenic tryptamine nucleoside phosphoramidates revealed that hHint3-1 and hHint3-2 are adenylate and phosphoramidate hydrolases with apparent second-order rate constants (k(cat)/K-m) ranging from 10(2) to 10(6) s(-1) M-1. Unlike hHint1, hHint3-1 and hHint3-2 prefer AIPA over tryptamine adenosine phosphoramidate by factors of 33- and 16-fold, respectively. In general, hHint3s hydrolyze phosphoramidate 370- to 2000-fold less efficiently than hHint1. Substitution of the potential active-site nucleophile, His145, by Ala was shown to abolish the adenylate and phosphoramidate hydrolase activity for hHint3-1. However, 0.2-0.4% residual activity was observed for the H145A mutant of hHint3-2. Both hHint3-1 and hHint3-2 were found to hydrolyze lysyl-adenylate generated by human lysyl-tRNA synthetase (hLysRS) by proceeding through an adenylated protein intermediate. hLysRS-dependent labeling of hHint3-1 and hHint3-2 was found to depend on His145, which aligns with the His112 of the Hint1 active site. The extent of active-site His145-AMP labeling was shown to be similar to His112-AMP labeling of hHint1. In contrast to all previously characterized members of the histidine triad superfamily, which have been shown to exist exclusively as homodimers, wild type and the H145A of hHint3-1 were found to exist across a range of multimeric states, from dimers to octamers and even larger oligomers, while wild type and the H145A of hHint3-2 exist predominantly in a monomeric state. The differences in oligomeric state may be important in vivo, because unlike tetracysteine-tagged Hint1, which was found along linear arrays exclusively in the cytoplasm in transfected HeLa cells, tagged hHint3-1 and Hint3-2 were found as aggregates both in the cytosol and in the nucleus. Taken together, these results imply that while Hint3 and Hint1 prefer aminoacyl-adenylates as substrates and catalytically interact with aminoacyl-tRNA synthetases, the significant differences in phosphoramidase activity, oligomeric state, and cellular localization suggest that Hint3s should be placed in a distinct branch of the histidine triad superfamily. (C) 2007 Elsevier Ltd. All rights reserved.
机译：人类Hint3（hHint3）已被分类为组氨酸三联体核苷酸（Hint）结合蛋白亚家族的成员。虽然Hint1在真核生物和原核生物中都普遍表达，但Hint3仅在真核生物中发现。以前，我们的实验室已经表征并比较了hHint1和大肠杆菌hinT的氨酰基-腺苷酸和核苷氨基磷酸酯水解酶活性。在这项研究中，hHint3-1（Ala36）及其单核苷酸多态性hHint3-2（A36G变体）被克隆，过表达和纯化。用合成的荧光吲哚丙酸酰基-腺苷酸（AIPA）和一系列荧光色胺色胺核苷氨基磷酸酯进行稳态动力学研究表明，hHint3-1和hHint3-2是腺苷酸和氨基磷酸酯水解酶，具有明显的二级速率常数（k（cat ）/ Km），范围从10（2）到10（6）s（-1）M-1。与hHint1不同，hHint3-1和hHint3-2与AI相比，对色胺类胺类磷酸氨基磷酸酯的偏好分别高33倍和16倍。通常，hHint3s的水解效率比hHint1低370-2000倍。已显示，用Ala取代潜在的活性位点亲核试剂His145可以消除hHint3-1的腺苷酸和氨基磷酸酯水解酶活性。然而，对于hHint3-2的H145A突变体观察到0.2-0.4％的残留活性。发现hHint3-1和hHint3-2都通过进行腺苷酸化的蛋白中间体水解由人赖氨酰tRNA合成酶（hLysRS）生成的赖氨酰腺苷酸。发现hHint3-1和hHint3-2的hLysRS依赖性标记依赖于His145，该标记与Hint1活性位点的His112对齐。活性位点His145-AMP标记的程度显示类似于hHint1的His112-AMP标记。与之前已被证明仅以同二聚体存在的组氨酸三联体超家族的所有成员相反，hHint3-1的野生型和H145A被发现存在于从二聚体到八聚体甚至更大的多种多聚态中。寡聚物，而野生型和hHint3-2的H145A主要以单体状态存在。寡聚状态的差异可能在体内很重要，因为不像四半胱氨酸标记的Hint1，是沿着线性阵列仅在转染的HeLa细胞的细胞质中发现的，而标记的hHint3-1和Hint3-2在细胞质和细胞质中都是聚集体。在细胞核中。两者合计，这些结果表明，虽然Hint3和Hint1更喜欢以氨酰基-腺苷酸为底物并与氨酰基-tRNA合成酶催化相互作用，但磷酰胺酶活性，寡聚状态和细胞定位方面的显着差异表明，Hint3应放置在组氨酸三联体超家族。 （C）2007 Elsevier Ltd.保留所有权利。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号