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腾讯登录Nucleic Acids Res：GluRS的人工进化
| 导读 | 近期，耶鲁大学分子生物物理学与生物化学系（Department of Molecular Biophysics and Biochemistry）的Dieter Söll课题组成功地通过人工进化的方法，将细菌来源的谷氨酰tRNA合成酶（glutamyl-tRNA synthetase, GluRS）转变为谷氨酰胺酰tRNA合成酶（glutaminyl-tRNA synthetase, GlnRS）... |
近期，耶鲁大学分子生物物理学与生物化学系（Department of Molecular Biophysics and Biochemistry）的Dieter Söll课题组成功地通过人工进化的方法，将细菌来源的谷氨酰tRNA合成酶（glutamyl-tRNA synthetase, GluRS）转变为谷氨酰胺酰tRNA合成酶（glutaminyl-tRNA synthetase, GlnRS），证明了这两个合成酶在进化上具有很近的亲缘关系。
郭立涛（Li-Tao Guo）博士是这个课题的主要参与人，他扩展了随机突变技术的应用，增加了人们对蛋白质进化的认识，丰富了研究方法，更增进了对酶的催化机理的了解。该研究成果发表在最新一期的《核酸研究》（<em>Nucleic Acids Research</em>）网络版上。
氨基酸的进化伴随着一类叫做氨酰tRNA合成酶（aminoacyl-tRNA synthetase，aaRS）的蛋白的进化，由进化初期的几种氨基酸到现在的22种，构成了当今物种的多样性。22种氨基酸对应着21种aaRS，人类在发现这类蛋白的开始，就对其进化关系有着浓厚的兴趣， GluRS和GlnRS就是aaRS中的一种。
<p align="center"><img src="http://www.bioon.com/biology/UploadFiles/201206/2012061210205161.jpg" alt="" width="500" height="276" border="0" /></p>
<p align="center">
GluRS和GlnRS的结构对比 (图片<br/>来源：<em>Nuc. Acids Res.</em>由郭立涛博士提供)</p>
<p align="center"><!--more--></p>
在自然界中，GluRS和GlnRS在序列上和结构上高度相似，具有很近的亲缘关系，因此认为GlnRS是从GluRS进化而来。长期以来，科学家们一直对这两种酶的进化关系十分感兴趣，尝试着通过定点突变的方法互换它们之间的识别特异性，但结果并不令人满意。大肠杆菌GlnRS活性中心的22个氨基酸曾经被定点突变，也只获得了十分微弱的GluRS活性，大约是真正GluRS的千分之一，可见GluRS和GlnRS对各自底物的识别是极其精确而复杂的。这个问题变得越来越困难：GluRS和GlnRS是如何实现对底物的精确识别呢？
Dieter Söll课题组长期从事tRNA及aaRS的研究，这一问题也吸引了他们的兴趣。这个课题的主要参与人--郭立涛博士，从SIBS毕业后，在耶鲁大学一直从事蛋白质工程研究工作。郭立涛博士把人工进化的方法应用到GluRS和GlnRS进化关系的研究上，经过不懈努力，成功地将细菌来源的GluRS进化出具有GlnRS活性的突变体，此突变体酶只有7个氨基酸被突变掉，但体内试验中中证明，这个突变体可以支持GlnRS温度敏感型大肠杆菌菌株在42度下生长（42度下，此菌株中的GlnRS是没有活性的，细胞不能生长），真正成为了具有GlnRS功能的蛋白质。
<a href="http://dx.doi.org/10.1093/nar/gks507">doi: 10.1093/nar/gks507</a>
PMC:
PMID:
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<br/><strong>Rational design and directed evolution of a bacterial-type glutaminyl-tRNA synthetase precursor
</strong><br/>
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.
<br/>来源：生物谷
</div>
</div>
</div>
郭立涛（Li-Tao Guo）博士是这个课题的主要参与人，他扩展了随机突变技术的应用，增加了人们对蛋白质进化的认识，丰富了研究方法，更增进了对酶的催化机理的了解。该研究成果发表在最新一期的《核酸研究》（<em>Nucleic Acids Research</em>）网络版上。
氨基酸的进化伴随着一类叫做氨酰tRNA合成酶（aminoacyl-tRNA synthetase，aaRS）的蛋白的进化，由进化初期的几种氨基酸到现在的22种，构成了当今物种的多样性。22种氨基酸对应着21种aaRS，人类在发现这类蛋白的开始，就对其进化关系有着浓厚的兴趣， GluRS和GlnRS就是aaRS中的一种。
<p align="center"><img src="http://www.bioon.com/biology/UploadFiles/201206/2012061210205161.jpg" alt="" width="500" height="276" border="0" /></p>
<p align="center">
GluRS和GlnRS的结构对比 (图片<br/>来源：<em>Nuc. Acids Res.</em>由郭立涛博士提供)</p>
<p align="center"><!--more--></p>
在自然界中，GluRS和GlnRS在序列上和结构上高度相似，具有很近的亲缘关系，因此认为GlnRS是从GluRS进化而来。长期以来，科学家们一直对这两种酶的进化关系十分感兴趣，尝试着通过定点突变的方法互换它们之间的识别特异性，但结果并不令人满意。大肠杆菌GlnRS活性中心的22个氨基酸曾经被定点突变，也只获得了十分微弱的GluRS活性，大约是真正GluRS的千分之一，可见GluRS和GlnRS对各自底物的识别是极其精确而复杂的。这个问题变得越来越困难：GluRS和GlnRS是如何实现对底物的精确识别呢？
Dieter Söll课题组长期从事tRNA及aaRS的研究，这一问题也吸引了他们的兴趣。这个课题的主要参与人--郭立涛博士，从SIBS毕业后，在耶鲁大学一直从事蛋白质工程研究工作。郭立涛博士把人工进化的方法应用到GluRS和GlnRS进化关系的研究上，经过不懈努力，成功地将细菌来源的GluRS进化出具有GlnRS活性的突变体，此突变体酶只有7个氨基酸被突变掉，但体内试验中中证明，这个突变体可以支持GlnRS温度敏感型大肠杆菌菌株在42度下生长（42度下，此菌株中的GlnRS是没有活性的，细胞不能生长），真正成为了具有GlnRS功能的蛋白质。
<a href="http://dx.doi.org/10.1093/nar/gks507">doi: 10.1093/nar/gks507</a>
PMC:
PMID:
<div>
<div>
<div>
<br/><strong>Rational design and directed evolution of a bacterial-type glutaminyl-tRNA synthetase precursor
</strong><br/>
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.
<br/>来源：生物谷
</div>
</div>
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