近期，中科院上海生命科学研究员生物化学与细胞生物学研究所王恩多研究组在Biochemical Journal发表研究论文：抗广谱药物AN2690的真核亮氨酰-tRNA合成酶的转移后的编校功能。
该研究组已经揭示亮氨酰-tRNA合成酶(LeuRS)具有多条编校途径，其转移后编校结构域位于插入活性中心的长的连接肽链1 (Connective Peptide 1, CP1）结构域。美国Anacor制药公司筛选出一种编号为AN2690的广谱抗真菌药5-fluoro-1,3-dihydro-1-hydroxy-2,1- benzoxaborole，它使酵母LeuRS的氨基酰化活力下降,丧失转移后的编校。生物化学与结构生物学的研究证实，该化合物与位于酶的CP1结构域的tRNA的3’末端共价结合，其作用靶点是真菌LeuRS的CP1结构域(Science, 2007, 316, 1759-1761)。
王恩多实验室的周小龙博士基于真核来源的贾第虫LeuRS（GlLeuRS）的结构和功能的研究，研究和比较了GlLeuRS和人胞质LeuRS（hcLeuRS）存在于CP1中一个称为真核专一的插入片段1（Eukarya-Specific Insertion 1, ESI）的元件，发现ESI参与氨基酸活化、tRNA氨基酰化和转移后编校反应，但不影响转移后编校反应编校的特异性。研究揭示出GlLeuRS的转移后编校功能是其C-端的tRNA结合结构域和CP1结构域协同作用的结果。AN2690不影响GlLeuRS的tRNA亮氨酰化活力和转移后的编校活力，而影响hcLeuRS的这两种活力。这说明GlLeuRS与hcLeuRS的CP1结构域有细微的差异。在GlLeuRS的CP1结构域中引入关键的突变点则可以恢复AN290抑制两种活力的作用。
本篇工作的意义在于：发现LeuRS的结构在进化过程中逐渐变化，随之其功能也相应微调；GlLeuRS与hcLeuRS的CP1结构域的差异将为针对GlLeuRS的编校活性结构域为靶点设计治疗贾第虫病的专一药物提供依据，而该药物应该对人细胞无影响。
该研究得到中国科学院，科技部重大科学研究计划，国家自然科学基金委，上海市科委的资助。
生物谷推荐原文出处：
Biochemical Journal doi:10.1042/BJ20100474
Post-transfer editing by a eukaryotic leucyl-tRNA synthetase resistant to the broad-spectrum drug AN2690
XiaoLong Zhou, Min Tan, Meng Wang, Xin Chen and En?Duo Wang1
State Key Laboratory of Molecular Biology and Graduate School of the Chinese Academy of Sciences, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, The Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, People's Republic of China
Some aaRSs (aminoacyl-tRNA synthetases) develop editing mechanisms to correct mis-charged tRNA. The CP1 (connective peptide 1) domain of LeuRS (leucyl-tRNA synthetase) contains the editing active site, which is the proven target for the broad-spectrum drug AN2690 (5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole). The ESI (eukarya-specific insertion 1) in the CP1 domain of GlLeuRS (Giardia lamblia LeuRS) has been identified. Similar substitution with the ESI from HsLeuRS (Homo sapiens LeuRS) impeded the leucine activation, aminoacylation and post-transfer editing of the enzyme, but had no effect on the editing specificity toward non-specific amino acids. Thr341 in GlLeuRS served as a specificity discriminator, as found in other LeuRS systems, although its substitution with an alanine residue did not destroy Leu-tRNALeu synthesis in vitro and in vivo. The Arg338 was crucial for tRNALeu charging and the Asp440 was crucial for leucine activation and aminoacylation. The post-transfer editing required the CTD (C-terminal domain), Arg338 and Asp440 of GlLeuRS. Interestingly, GlLeuRS was completely resistant to the AN2690, which is an inhibitor of various LeuRSs. The universally conserved aspartate residue in the LeuRS CP1 domains was responsible for the resistance of GlLeuRS and another recently reported AN2690-resistant AaLeuRS (Aquifex aeolicus LeuRS). Our results indicate the functional divergence of some absolutely conserved sites, improve the understanding of the editing function of eukaryotic/archaeal LeuRSs and shed light on the development of a GlLeuRS-specific inhibitor for the treatment of giardiasis.
