8C49

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

Summary for 8C49

• Entry DOI: 10.2210/pdb8c49/pdb
• Descriptor: Pyrrolysyl-tRNA synthetase, PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER, DI(HYDROXYETHYL)ETHER, ... (7 entities in total)
• Functional Keywords: pyrrolysyl-trna synthetase, genetic code expansion, engineered translation components, methanomethylophilus alvus, ligase
• Biological source: Candidatus Methanomethylophilus alvus
• Total number of polymer chains: 2
• Total formula weight: 66439.65

Authors

Hardy, F.J.,Levy, C.W. (deposition date: 2023-01-03, release date: 2023-07-19, Last modification date: 2024-02-07)

Primary citation

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

Experimental method

X-RAY DIFFRACTION (1.82 Å)