8C49

Crystal structure of pyrrolysyl-tRNA synthetase from Methanomethylophilus alvus engineered for 3-Methyl-L-histidine, bound to AMPPNP

8C49 の概要

• エントリーDOI: 10.2210/pdb8c49/pdb
• 分子名称: Pyrrolysyl-tRNA synthetase, PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER, DI(HYDROXYETHYL)ETHER, ... (7 entities in total)
• 機能のキーワード: pyrrolysyl-trna synthetase, genetic code expansion, engineered translation components, methanomethylophilus alvus, ligase
• 由来する生物種: Candidatus Methanomethylophilus alvus
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 66439.65

構造登録者

Hardy, F.J.,Levy, C.W. (登録日: 2023-01-03, 公開日: 2023-07-19, 最終更新日: 2024-02-07)

主引用文献

Taylor, C.J.,Hardy, F.J.,Burke, A.J.,Bednar, R.M.,Mehl, R.A.,Green, A.P.,Lovelock, S.L.
Engineering mutually orthogonal PylRS/tRNA pairs for dual encoding of functional histidine analogues.
Protein Sci., 32:e4640-e4640, 2023

PubMed Abstract: 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.

PubMed: 37051694
DOI: 10.1002/pro.4640

実験手法

X-RAY DIFFRACTION (1.82 Å)