美國(guó)康涅狄格州 電 /生物谷BIOON/ 2012年6月12日 -- 近期,，耶魯大學(xué)分子生物物理學(xué)與生物化學(xué)系（Department of Molecular Biophysics and Biochemistry）的Dieter Söll課題組成功地通過(guò)人工進(jìn)化的方法,，將細(xì)菌來(lái)源的谷氨酰tRNA合成酶（glutamyl-tRNA synthetase, GluRS）轉(zhuǎn)變?yōu)楣劝滨０孵RNA合成酶（glutaminyl-tRNA synthetase, GlnRS），證明了這兩個(gè)合成酶在進(jìn)化上具有很近的親緣關(guān)系,。
郭立濤（Li-Tao Guo）博士是這個(gè)課題的主要參與人,，他擴(kuò)展了隨機(jī)突變技術(shù)的應(yīng)用，增加了人們對(duì)蛋白質(zhì)進(jìn)化的認(rèn)識(shí),，豐富了研究方法,，更增進(jìn)了對(duì)酶的催化機(jī)理的了解。該研究成果發(fā)表在最新一期的《核酸研究》（Nucleic Acids Research）網(wǎng)絡(luò)版上,。
氨基酸的進(jìn)化伴隨著一類(lèi)叫做氨酰tRNA合成酶（aminoacyl-tRNA synthetase,，aaRS）的蛋白的進(jìn)化，由進(jìn)化初期的幾種氨基酸到現(xiàn)在的22種,，構(gòu)成了當(dāng)今物種的多樣性,。22種氨基酸對(duì)應(yīng)著21種aaRS，人類(lèi)在發(fā)現(xiàn)這類(lèi)蛋白的開(kāi)始,，就對(duì)其進(jìn)化關(guān)系有著濃厚的興趣,， GluRS和GlnRS就是aaRS中的一種。
GluRS和GlnRS的結(jié)構(gòu)對(duì)比 (圖片來(lái)源：Nuc. Acids Res.由郭立濤博士提供)
在自然界中,，GluRS和GlnRS在序列上和結(jié)構(gòu)上高度相似,，具有很近的親緣關(guān)系，因此認(rèn)為GlnRS是從GluRS進(jìn)化而來(lái),。長(zhǎng)期以來(lái),，科學(xué)家們一直對(duì)這兩種酶的進(jìn)化關(guān)系十分感興趣，嘗試著通過(guò)定點(diǎn)突變的方法互換它們之間的識(shí)別特異性,，但結(jié)果并不令人滿(mǎn)意,。大腸桿菌GlnRS活性中心的22個(gè)氨基酸曾經(jīng)被定點(diǎn)突變，也只獲得了十分微弱的GluRS活性,，大約是真正GluRS的千分之一,，可見(jiàn)GluRS和GlnRS對(duì)各自底物的識(shí)別是極其精確而復(fù)雜的。這個(gè)問(wèn)題變得越來(lái)越困難：GluRS和GlnRS是如何實(shí)現(xiàn)對(duì)底物的精確識(shí)別呢,？
Dieter Söll課題組長(zhǎng)期從事tRNA及aaRS的研究,，這一問(wèn)題也吸引了他們的興趣。這個(gè)課題的主要參與人--郭立濤博士,，從SIBS畢業(yè)后,，在耶魯大學(xué)一直從事蛋白質(zhì)工程研究工作。郭立濤博士把人工進(jìn)化的方法應(yīng)用到GluRS和GlnRS進(jìn)化關(guān)系的研究上,，經(jīng)過(guò)不懈努力,，成功地將細(xì)菌來(lái)源的GluRS進(jìn)化出具有GlnRS活性的突變體，此突變體酶只有7個(gè)氨基酸被突變掉,，但體內(nèi)試驗(yàn)中中證明,，這個(gè)突變體可以支持GlnRS溫度敏感型大腸桿菌菌株在42度下生長(zhǎng)（42度下,，此菌株中的GlnRS是沒(méi)有活性的，細(xì)胞不能生長(zhǎng)）,，真正成為了具有GlnRS功能的蛋白質(zhì),。（生物谷Bioon.com）
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doi: 10.1093/nar/gks507
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Rational design and directed evolution of a bacterial-type glutaminyl-tRNA synthetase precursor
Li-Tao Guo, Sunna Helgadóttir, Dieter Söll,and Jiqiang Ling
Protein biosynthesis requires aminoacyl-transfer RNA (tRNA) synthetases to provide aminoacyl-tRNA substrates for the ribosome. Most bacteria and all archaea lack a glutaminyl-tRNA synthetase (GlnRS); instead, Gln-tRNAGln is produced via an indirect pathway: a glutamyl-tRNA synthetase (GluRS) first attaches glutamate (Glu) to tRNAGln, and an amidotransferase converts Glu-tRNAGln to Gln-tRNAGln. The human pathogen Helicobacter pylori encodes two GluRS enzymes, with GluRS2 specifically aminoacylating Glu onto tRNAGln. It was proposed that GluRS2 is evolving into a bacterial-type GlnRS. Herein, we have combined rational design and directed evolution approaches to test this hypothesis. We show that, in contrast to wild-type (WT) GlnRS2, an engineered enzyme variant (M110) with seven amino acid changes is able to rescue growth of the temperature-sensitive Escherichia coli glnS strain UT172 at its non-permissive temperature. In vitro kinetic analyses reveal that WT GluRS2 selectively acylates Glu over Gln, whereas M110 acylates Gln 4-fold more efficiently than Glu. In addition, M110 hydrolyzes adenosine triphosphate 2.5-fold faster in the presence of Glu than Gln, suggesting that an editing activity has evolved in this variant to discriminate against Glu. These data imply that GluRS2 is a few steps away from evolving into a GlnRS and provides a paradigm for studying aminoacyl-tRNA synthetase evolution using directed engineering approaches.
