Li Wen-Tai, Mahapatra Anirban, Longstaff David G, Bechtel Jonathan, Zhao Gang, Kang Patrick T, Chan Michael K, Krzycki Joseph A
Department of Chemistry, The Ohio State University, Columbus, OH 43210, USA.
J Mol Biol. 2009 Jan 30;385(4):1156-64. doi: 10.1016/j.jmb.2008.11.032. Epub 2008 Nov 25.
Pyrrolysine, the 22nd amino acid, is encoded by amber (TAG=UAG) codons in certain methanogenic archaea and bacteria. PylS, the pyrrolysyl-tRNA synthetase, ligates pyrrolysine to tRNA(Pyl) for amber decoding as pyrrolysine. PylS and tRNA(Pyl) have potential utility in making tailored recombinant proteins. Here, we probed interactions necessary for recognition of substrates by archaeal PylS via synthesis of close pyrrolysine analogs and testing their reactivity in amino acid activation assays. Replacement of the methylpyrroline ring of pyrrolysine with cyclopentane indicated that solely hydrophobic interactions with the ring-binding pocket of PylS are sufficient for substrate recognition. However, a 100-fold increase in the specificity constant of PylS was observed with an analog, 2-amino-6-((R)-tetrahydrofuran-2-carboxamido)hexanoic acid (2Thf-lys), in which tetrahydrofuran replaced the pyrrolysine methylpyrroline ring. Other analogs in which the electronegative atom was moved to different positions suggested PylS preference for a hydrogen-bond-accepting group at the imine nitrogen position in pyrrolysine. 2Thf-lys was a preferred substrate over a commonly employed pyrrolysine analog, but the specificity constant for 2Thf-lys was 10-fold lower than for pyrrolysine itself, largely due to the change in K(m). The in vivo activity of the analogs in supporting UAG suppression in Escherichia coli bearing genes for PylS and tRNA(Pyl) was similar to in vitro results, with L-pyrrolysine and 2Thf-lys supporting the highest amounts of UAG translation. Increasing concentrations of either PylS substrate resulted in a linear increase in UAG suppression, providing a facile method to assay bioactive pyrrolysine analogs. These results illustrate the relative importance of the H-bonding and hydrophobic interactions in the recognition of the methylpyrroline ring of pyrrolysine and provide a promising new series of easily synthesized pyrrolysine analogs that can serve as scaffolds for the introduction of novel functional groups into recombinant proteins.
吡咯赖氨酸是第22种氨基酸，在某些产甲烷古菌和细菌中由琥珀密码子（TAG = UAG）编码。吡咯赖氨酰 - tRNA合成酶（PylS）将吡咯赖氨酸连接到tRNA(Pyl)上，用于将琥珀密码子解码为吡咯赖氨酸。PylS和tRNA(Pyl)在制备定制重组蛋白方面具有潜在用途。在这里，我们通过合成吡咯赖氨酸的紧密类似物并在氨基酸活化试验中测试它们的反应性，探究了古菌PylS识别底物所需的相互作用。用环戊烷取代吡咯赖氨酸的甲基吡咯啉环表明，仅与PylS的环结合口袋的疏水相互作用就足以实现底物识别。然而，观察到一种类似物2-氨基-6-((R)-四氢呋喃-2-甲酰胺基)己酸（2Thf-lys）使PylS的特异性常数增加了100倍，其中四氢呋喃取代了吡咯赖氨酸的甲基吡咯啉环。其他将电负性原子移至不同位置的类似物表明，PylS更倾向于吡咯赖氨酸中亚胺氮位置上的氢键接受基团。2Thf-lys比常用的吡咯赖氨酸类似物更受青睐，但2Thf-lys的特异性常数比吡咯赖氨酸本身低10倍，这主要是由于K(m)的变化。这些类似物在携带PylS和tRNA(Pyl)基因的大肠杆菌中支持UAG抑制的体内活性与体外结果相似，L-吡咯赖氨酸和2Thf-lys支持的UAG翻译量最高。增加任何一种PylS底物的浓度都会导致UAG抑制呈线性增加，这为检测生物活性吡咯赖氨酸类似物提供了一种简便方法。这些结果说明了氢键和疏水相互作用在识别吡咯赖氨酸甲基吡咯啉环中的相对重要性，并提供了一系列有前景的易于合成的吡咯赖氨酸类似物，它们可作为将新官能团引入重组蛋白的支架。
