近期,，中科院上海生命科學(xué)研究員生物化學(xué)與細(xì)胞生物學(xué)研究所王恩多研究組在Biochemical Journal發(fā)表研究論文：抗廣譜藥物AN2690的真核亮氨酰-tRNA合成酶的轉(zhuǎn)移后的編校功能。
該研究組已經(jīng)揭示亮氨酰-tRNA合成酶(LeuRS)具有多條編校途徑，其轉(zhuǎn)移后編校結(jié)構(gòu)域位于插入活性中心的長(zhǎng)的連接肽鏈1 (Connective Peptide 1, CP1）結(jié)構(gòu)域,。美國(guó)Anacor制藥公司篩選出一種編號(hào)為AN2690的廣譜抗真菌藥5-fluoro-1,3-dihydro-1-hydroxy-2,1- benzoxaborole,，它使酵母LeuRS的氨基?；盍ο陆?喪失轉(zhuǎn)移后的編校,。生物化學(xué)與結(jié)構(gòu)生物學(xué)的研究證實(shí)，該化合物與位于酶的CP1結(jié)構(gòu)域的tRNA的3’末端共價(jià)結(jié)合,，其作用靶點(diǎn)是真菌LeuRS的CP1結(jié)構(gòu)域(Science, 2007, 316, 1759-1761),。
王恩多實(shí)驗(yàn)室的周小龍博士基于真核來(lái)源的賈第蟲(chóng)LeuRS（GlLeuRS）的結(jié)構(gòu)和功能的研究，研究和比較了GlLeuRS和人胞質(zhì)LeuRS（hcLeuRS）存在于CP1中一個(gè)稱(chēng)為真核專(zhuān)一的插入片段1（Eukarya-Specific Insertion 1, ESI）的元件,，發(fā)現(xiàn)ESI參與氨基酸活化,、tRNA氨基酰化和轉(zhuǎn)移后編校反應(yīng),，但不影響轉(zhuǎn)移后編校反應(yīng)編校的特異性,。研究揭示出GlLeuRS的轉(zhuǎn)移后編校功能是其C-端的tRNA結(jié)合結(jié)構(gòu)域和CP1結(jié)構(gòu)域協(xié)同作用的結(jié)果。AN2690不影響GlLeuRS的tRNA亮氨?；盍娃D(zhuǎn)移后的編校活力,，而影響hcLeuRS的這兩種活力,。這說(shuō)明GlLeuRS與hcLeuRS的CP1結(jié)構(gòu)域有細(xì)微的差異。在GlLeuRS的CP1結(jié)構(gòu)域中引入關(guān)鍵的突變點(diǎn)則可以恢復(fù)AN290抑制兩種活力的作用。
本篇工作的意義在于：發(fā)現(xiàn)LeuRS的結(jié)構(gòu)在進(jìn)化過(guò)程中逐漸變化,，隨之其功能也相應(yīng)微調(diào),；GlLeuRS與hcLeuRS的CP1結(jié)構(gòu)域的差異將為針對(duì)GlLeuRS的編校活性結(jié)構(gòu)域?yàn)榘悬c(diǎn)設(shè)計(jì)治療賈第蟲(chóng)病的專(zhuān)一藥物提供依據(jù),，而該藥物應(yīng)該對(duì)人細(xì)胞無(wú)影響,。
該研究得到中國(guó)科學(xué)院，科技部重大科學(xué)研究計(jì)劃,，國(guó)家自然科學(xué)基金委,，上海市科委的資助。（生物谷Bioon.com）
生物谷推薦原文出處：
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.
