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Publication Abstract from PubMed

Pairs of pyrrolysyl-tRNA synthetase (PylRS) and tRNA(Pyl) from Methanosarcina mazei and Methanosarcina barkeri 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 N(epsilon)-((((E)-cyclooct-2-en-1-yl)oxy)carbonyl)-L-lysine (TCO*Lys), by using Methanomethylophilus alvus PylRS with structure-based mutations in and around the amino acid binding pocket (first-layer and second-layer mutations, respectively). Recently, the PylRS.tRNA(Pyl) 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 N(epsilon)-(p-ethynylbenzyloxycarbonyl)-L-lysine (pEtZLys) into proteins, but it was much less efficient than that of TCO*Lys with M. alvus 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 pEtZLys up to 57 +/- 8% of that with TCO*Lys at high enzyme concentrations in the cell-free protein synthesis.

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.,Yanagisawa T, Seki E, Tanabe H, Fujii Y, Sakamoto K, Yokoyama S Int J Mol Sci. 2023 Mar 26;24(7):6256. doi: 10.3390/ijms24076256. PMID:37047230^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.