Recognition of Non-α-amino Substrates by Pyrrolysyl-tRNA Synthetase
Recognition of Non-α-amino Substrates by Pyrrolysyl-tRNA Synthetase
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DOI：
10.1016/j.jmb.2008.11.059
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发表时间：
2009-02-06
影响因子：
5.6
通讯作者：
Yokoyama, Shigeyuki
中科院分区：
文献类型：
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作者：
Kobayashi, Takatsugu;Yanagisawa, Tatsuo;Yokoyama, Shigeyuki
吡咯赖氨酰-tRNA合成酶（PylRS）是最近在一些产甲烷古菌和细菌中发现的氨酰-tRNA合成酶（阿尔斯），其识别异常大的赖氨酸衍生物L-吡咯赖氨酸作为底物，并将其附着到同源tRNA（tRNA（Pyl））上。PylRS-tRNA（Pyl）对不与大肠杆菌中的内源性aaRS-tRNA对相互作用，因此可以用作用于遗传密码扩增的新型aaRS-tRNA对。Mazei甲烷八叠球菌PylRS的晶体结构显示，它具有一个独特的、大的氨基酸结合口袋，而野生型M. Mazei PylRS识别天然赖氨酸衍生物以及许多赖氨酸类似物，包括具有不同侧链大小和结构的N-叔丁氧羰基-L-赖氨酸（Boc-赖氨酸）。此外，PylRS仅松散地识别底物的α-氨基，而大多数aaRS，包括结构上和遗传上相关的苯丙氨酰-tRNA合成酶（PheRS），严格地识别底物的主链基团。我们在此报道野生型PylRS可以识别具有多种主链α-基团的底物：α-羟基酸、非α-氨基羧酸、N-α-甲基氨基酸和D-氨基酸，每种都具有与Boc-赖氨酸相同的侧链。相比之下，PheRS不识别这些氨基酸类似物。通过在大肠杆菌中表达野生型PylRS及其同源tRNA（Pyl），在Boc-赖氨酸的α-羟基酸类似物（Boc-LysOH）的存在下，将琥珀密码子（UAG）成功地重新编码为Boc-LysOH，从而将酯键位点特异性地掺入蛋白质分子中。该PylRS-tRNA（Pyl）对预期扩大通过体内和体外核糖体翻译产生的蛋白质分子的主链多样性。(C)2008爱思唯尔有限公司保留所有权利。
Pyrrolysyl-tRNA synthetase (PylRS), an aminoacyl-tRNA synthetase (aaRS) recently found in some methanogenic archaea and bacteria, recognizes an unusually large lysine derivative, L-pyrrolysine, as the substrate, and attaches it to the cognate tRNA (tRNA(Pyl)). The PylRS-tRNA(Pyl) pair interacts with none of the endogenous aaRS-tRNA pairs in Escherichia coli, and thus can be used as a novel aaRS-tRNA pair for genetic code expansion. The crystal structures of the Methanosarcina mazei PylRS revealed that it has a unique, large pocket for amino acid binding, and the wild type M. mazei PylRS recognizes the natural lysine derivative as well as many lysine analogs, including N-epsilon-(tert-butoxycarbonyl)-L-lysine (Boc-lysine), with diverse side chain sizes and structures. Moreover, the PylRS only loosely recognizes the alpha-amino group of the substrate, whereas most aaRSs, including the structurally and geneticatly related phenylalanyl-tRNA synthetase (PheRS), strictly recognize the main chain groups of the substrate. We report here that wild type PylRS can recognize substrates with a variety of main-chain a-groups: alpha-hydroxyacid, non-alpha-amino-carboxylic acid, N-alpha-methyl-amino acid, and D-amino acid, each with the same side chain as that of Boc-lysine. In contrast, PheRS recognizes none of these amino acid analogs. By expressing the wild type PylRS and its cognate tRNA(Pyl) in E. coli in the presence of the alpha-hydroxyacid analog of Boc-lysine (Boc-LysOH), the amber codon (UAG) was recoded successfully as Boc-LysOH, and thus an ester bond was site-specifically incorporated into a protein molecule. This PylRS-tRNA(Pyl) pair is expected to expand the backbone diversity of protein molecules produced by both in vivo and in vitro ribosomal translation. (C) 2008 Elsevier Ltd. All rights reserved.