The Pyrrolysyl-tRNA Synthetase Activity can be Improved by a P188 Mutation that Stabilizes the Full-Length Enzyme.
The Pyrrolysyl-tRNA Synthetase Activity can be Improved by a P188 Mutation that Stabilizes the Full-Length Enzyme.
复制标题
DOI：
10.1016/j.jmb.2022.167453
复制
发表时间：
2022-04-30
影响因子：
5.6
通讯作者：
Liu, Wenshe
中科院分区：
文献类型：
--
作者：
Cho, Chia-Chuan;Blankenship, Lauren R.;Ma, Xinyu;Xu, Shiqing;Liu, Wenshe
关键词：
基于琥珀抑制的非规范氨基酸突变技术在基础和应用研究中得到了广泛的应用。到目前为止，已经在原核生物和真核生物中使用野生型和工程化的吡咯赖氨酰-tRNA合成酶（PylRS）-tRNAPyl（PylT）对通过琥珀密码子遗传编码了超过200个ncAA。马氏甲烷八叠球菌PylRS（MmPylRS）可以说是两种最常用的PylRS变体之一。然而，它含有一个不稳定的N-末端结构域，该结构域通常在表达过程中从全长蛋白质上切割下来，因此导致酶活性低。我们发现切割发生在A189之后，并且当MmPylRS与Ca共表达时，这种切割被抑制。嗜甲烷甲基菌tRNAPyl（CmaPylT）。在CmaPylT的存在下，MmPylRS在替代位点K110之后被切割。MmPylRS对CmaPylT有活性。其与CmaPylT的组合使用导致在琥珀密码子处的Nε-Boc-赖氨酸（BocK）的掺入增强。为了防止MmPylRS在其同源M存在下在A189之后裂解。Mazei tRNAPyl（MmPylT），我们在P188处引入突变。我们的结果表明P188 G突变稳定了MmPylRS。我们表明，野生型MmPylRS或其工程化变体中的P188 G突变允许BocK和其他非规范氨基酸（包括Nε-乙酰基-赖氨酸）在与MmPylT共表达时增强的掺入。
The amber suppression-based noncanonical amino acid (ncAA) mutagenesis technique has been widely used in both basic and applied research. So far more than two hundred ncAAs have been genetically encoded by amber codon in both prokaryotes and eukaryotes using wild-type and engineered pyrrolysyl-tRNA synthetase (PylRS)-tRNAPyl (PylT) pairs. Methanosarcina mazei PylRS (MmPylRS) is arguably one of two most used PylRS variants. However, it contains an unstable N-terminal domain that is usually cleaved from the full-length protein during expression and therefore leads to a low enzyme activity. We discovered that the cleavage takes place after A189 and this cleavage is inhibited when MmPylRS is co-expressed with Ca. Methanomethylophilus alvus tRNAPyl (CmaPylT). In the presence of CmaPylT, MmPylRS is cleaved after an alternative site K110. MmPylRS is active toward CmaPylT. Its combined use with CmaPylT leads to enhanced incorporation of Nε-Boc-lysine (BocK) at amber codon. To prevent MmPylRS from cleavage after A189 in the presence of its cognate M. mazei tRNAPyl (MmPylT), we introduced mutations at P188. Our results indicated that the P188G mutation stabilizes MmPylRS. We showed that the P188G mutation in wild-type MmPylRS or its engineered variants allows enhanced incorporation of BocK and other noncanonical amino acids including Nε-acetyl-lysine when they are co-expressed with MmPylT.
登录
查看更多内容
影响因子：
16.6
作者：
Chen, Peng R.;Groff, Dan;Guo, Jiantao;Ou, Weijia;Cellitti, Susan;Geierstanger, Bernhard H.;Schultz, Peter G.
通讯作者：
Schultz, Peter G.
影响因子：
4
作者：
Lee, Yan-Jiun;Wu, Bo;Raymond, Jeffrey E.;Zeng, Yu;Fang, Xinqiang;Wooley, Karen L.;Liu, Wenshe R.
通讯作者：
Liu, Wenshe R.
影响因子：
4.8
作者：
Jiang, Ruisheng;Krzycki, Joseph A.
通讯作者：
Krzycki, Joseph A.
影响因子：
2.7
作者：
Jiang, XY;Zhang, MY;Muramatu, M
通讯作者：
Muramatu, M
影响因子：
46.9
作者：
Pédelacq, JD;Cabantous, S;Waldo, GS
通讯作者：
Waldo, GS