8IFJ

Crystal structure of pyrrolysyl-tRNA synthetase from methanogenic archaeon ISO4-G1

Summary for 8IFJ

• Entry DOI: 10.2210/pdb8ifj/pdb
• Descriptor: Pyrrolysyl-tRNA synthetase PylS (2 entities in total)
• Functional Keywords: non-canonical amino acids, genetic code expansion, trna, translation
• Biological source: methanogenic archaeon mixed culture ISO4-G1
• Total number of polymer chains: 10
• Total formula weight: 312564.69

Authors

Yanagisawa, T.,Tanabe, H.,Yokoyama, S. (deposition date: 2023-02-18, release date: 2023-03-15, Last modification date: 2024-05-29)

Primary citation

Yanagisawa, T.,Seki, E.,Tanabe, H.,Fujii, Y.,Sakamoto, K.,Yokoyama, S.
Crystal Structure of Pyrrolysyl-tRNA Synthetase from a Methanogenic Archaeon ISO4-G1 and Its Structure-Based Engineering for Highly-Productive Cell-Free Genetic Code Expansion with Non-Canonical Amino Acids.
Int J Mol Sci, 24:-, 2023

PubMed Abstract: Pairs of pyrrolysyl-tRNA synthetase (PylRS) and tRNA from and are widely used for site-specific incorporations of non-canonical amino acids into proteins (genetic code expansion). Previously, we achieved full productivity of cell-free protein synthesis for bulky non-canonical amino acids, including -(((()-cyclooct-2-en-1-yl)oxy)carbonyl)-L-lysine (TCO*Lys), by using PylRS with structure-based mutations in and around the amino acid binding pocket (first-layer and second-layer mutations, respectively). Recently, the PylRS·tRNA pair from a methanogenic archaeon ISO4-G1 was used for genetic code expansion. In the present study, we determined the crystal structure of the methanogenic archaeon ISO4-G1 PylRS (ISO4-G1 PylRS) and compared it with those of structure-known PylRSs. Based on the ISO4-G1 PylRS structure, we attempted the site-specific incorporation of -(-ethynylbenzyloxycarbonyl)-L-lysine (EtZLys) into proteins, but it was much less efficient than that of TCO*Lys with PylRS mutants. Thus, the first-layer mutations (Y125A and M128L) of ISO4-G1 PylRS, with no additional second-layer mutations, increased the protein productivity with EtZLys up to 57 ± 8% of that with TCO*Lys at high enzyme concentrations in the cell-free protein synthesis.

PubMed: 37047230
DOI: 10.3390/ijms24076256

Experimental method

X-RAY DIFFRACTION (2.78 Å)