Engineering mutually orthogonal PylRS/tRNA pairs for dual encoding of functional histidine analogues.
Engineering mutually orthogonal PylRS/tRNA pairs for dual encoding of functional histidine analogues.
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DOI：
10.1002/pro.4640
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发表时间：
2023-05
期刊：
影响因子：
8
通讯作者：
Lovelock, Sarah L. L.
中科院分区：
文献类型：
--
作者：
Taylor, Christopher J. J.;Hardy, Florence J. J.;Burke, Ashleigh J. J.;Bednar, Riley M. M.;Mehl, Ryan A. A.;Green, Anthony P. P.;Lovelock, Sarah L. L.
扩展遗传密码的可用性为酶设计和工程带来了令人兴奋的新机遇。在这方面，组氨酸类似物已被证明特别通用，可用作增强金属酶功能的配体和设计酶中的催化亲核试剂。基因编码多个功能残基的能力可以极大地扩展酶活性位点内可访问的化学范围。在这里，我们开发了相互正交的翻译组件来选择性编码两个结构相似的组氨酸类似物。将混杂的马氏甲烷八叠球菌吡咯赖氨酰-tRNA 合成酶 (MmPylRSIFGFF) 的已知突变移植到嗜甲烷菌 (MaPylRSIFGFF) 的单域 PylRS 中，提供了一种具有更高效率和 3-甲基-L-组氨酸 (MeHis) 掺入特异性的变体。使用体外生化测定和 X 射线晶体学进一步表征 MaPylRSIFGFF 克隆。随后，我们设计了正交 MmPylRS，以提高 3-(3-吡啶基)-L-丙氨酸 (3-Pyr) 的活性和选择性，将其与 MaPylRSIFGFF 结合使用，产生同时含有 3-Pyr 和 MeHis 的蛋白质。鉴于组氨酸在酶机制中发挥的多种作用，我们预计本研究中开发的工具将支持具有新功能和增强功能的酶的开发。
The availability of an expanded genetic code opens exciting new opportunities in enzyme design and engineering. In this regard histidine analogues have proven particularly versatile, serving as ligands to augment metalloenzyme function and as catalytic nucleophiles in designed enzymes. The ability to genetically encode multiple functional residues could greatly expand the range of chemistry accessible within enzyme active sites. Here, we develop mutually orthogonal translation components to selectively encode two structurally similar histidine analogues. Transplanting known mutations from a promiscuous Methanosarcina mazei pyrrolysyl‐tRNA synthetase (MmPylRSIFGFF) into a single domain PylRS from Methanomethylophilus alvus (MaPylRSIFGFF) provided a variant with improved efficiency and specificity for 3‐methyl‐L‐histidine (MeHis) incorporation. The MaPylRSIFGFF clone was further characterized using in vitro biochemical assays and x‐ray crystallography. We subsequently engineered the orthogonal MmPylRS for activity and selectivity for 3‐(3‐pyridyl)‐L‐alanine (3‐Pyr), which was used in combination with MaPylRSIFGFF to produce proteins containing both 3‐Pyr and MeHis. Given the versatile roles played by histidine in enzyme mechanisms, we anticipate that the tools developed within this study will underpin the development of enzymes with new and enhanced functions.
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