Manchester Institute of Biotechnology, School of Chemistry, University of Manchester, Manchester, UK.
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon, USA.
Protein Sci. 2023 May;32(5):e4640. doi: 10.1002/pro.4640.
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 (MmPylRS ) into a single domain PylRS from Methanomethylophilus alvus (MaPylRS ) provided a variant with improved efficiency and specificity for 3-methyl-L-histidine (MeHis) incorporation. The MaPylRS 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 MaPylRS 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.
扩展遗传密码的可用性为酶设计和工程开辟了令人兴奋的新机会。在这方面，组氨酸类似物已被证明特别多才多艺，它们可以作为配体来增强金属酶的功能，也可以作为设计酶中的催化亲核试剂。能够遗传编码多种功能残基可以大大扩展酶活性位点中可获得的化学范围。在这里，我们开发了相互正交的翻译组件来选择性地编码两种结构相似的组氨酸类似物。将已知的突变从一种混杂的 Methanosarcina mazei 吡咯赖氨酸-tRNA 合成酶（MmPylRS）移植到来自 Methanomethylophilus alvus 的单个结构域 PylRS（MaPylRS）中，提供了一种对 3-甲基-L-组氨酸（MeHis）掺入具有更高效率和特异性的变体。使用体外生化测定和 X 射线晶体学进一步表征了 MaPylRS 克隆。随后，我们对正交的 MmPylRS 进行了活性和 3-(3-吡啶基)-L-丙氨酸（3-Pyr）选择性的工程改造，与 MaPylRS 一起用于产生同时含有 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为开发具有新功能和增强功能的酶提供基础。
