Altering the Enantioselectivity of Tyrosyl-tRNA Synthetase by Insertion of a Stereospecific Editing Domain

Abstract

Translation of mRNAs by the ribosome is stereospecific, with only l-amino acids being incorporated into the nascent polypeptide chain. This stereospecificity results from the exclusion of d-amino acids at three steps during protein synthesis: (1) the aminoacylation of tRNA by aminoacyl-tRNA synthetases, (2) binding of aminoacyl-tRNAs to EF-Tu, and (3) recognition of aminoacyl-tRNAs by the ribosome. As a first step toward incorporating d-amino acids during protein synthesis, we have altered the enantioselectivity of tyrosyl-tRNA synthetase. This enzyme is unusual among aminoacyl-tRNA synthetases, as it can aminoacylate tRNA with d-tyrosine (albeit at a reduced rate compared to l-tyrosine). To change the enantioselectivity of tyrosyl-tRNA synthetase, we introduced the post-transfer editing domain from Pyrococcus horikoshii phenylalanyl-tRNA synthetase into the connective polypeptide 1 (CP1) domain of Geobacillus stearothermophilus tyrosyl-tRNA synthetase (henceforth designated TyrRS-FRSed). We show that the phenylalanyl-tRNA synthetase editing domain is stereospecific, hydrolyzing l-Tyr-tRNA(Tyr), but not d-Tyr-tRNA(Tyr). We further show that inserting the phenylalanyl-tRNA synthetase editing domain into the CP1 domain of tyrosyl-tRNA synthetase decreases the activity of the synthetic site in tyrosyl-tRNA synthetase. This decrease in activity is critical, as it prevents the rate of synthesis from overwhelming the ability of the editing domain to hydrolyze the l-Tyr-tRNA(Tyr) product. Overall, inserting the phenylalanyl-tRNA synthetase editing domain results in a 2-fold shift in the enantioselectivity of tyrosyl-tRNA synthetase toward the d-Tyr-tRNA(Tyr) product. When a 4-fold excess of d-tyrosine is used, approximately 40% of the tRNA(Tyr) is aminoacylated with d-tyrosine.

Mechanistic insights into the slow peptide bond formation with D-amino acids in the ribosomal active site

“…Over the past years, messenger RNA-dependent synthesis of D-amino acid-containing proteins became possible in vitro via engineering of different translation machinery components. For example, protein engineering allowed the creation of aminoacyl-tRNA synthetases that selectively use D-isomer of tyrosine (25,26). Also, development of engineered catalytic RNAs, flexizymes, made possible production of D-aminoacyl-tRNAs for use in cell-free protein translation systems (27).…”

Plasticity and Constraints of tRNA Aminoacylation Define Directed Evolution of Aminoacyl-tRNA Synthetases

“…Interestingly, this editing domain of archaeal PheRS also shows stereospecificity, and hydrolyzes l -Tyr-tRNA, but not d -Tyr-tRNA. A TyrRS-PheRS chimera has been shown to possess increased enantioselectivity toward d -Tyr-tRNA Tyr [35]. A similar approach was used to design a human mitochondrial PheRS variant that is “resistant” to misacylation of the reactive oxygen species (ROS) derived from Phe (such as Tyr and m -Tyr).…”

The central role of tRNA in genetic code expansion

“…To enhance production of the 3-I-Tyr-tRNA Tyr and to clear Tyr-tRNA Tyr , the editing domain of the P. horikoshii PheRS was inserted into iodoTyrRS, creating a variant (iodoTyrRS-ed) that was able to efficiently produced 3-iodo- L -tyrosine-tRNA Tyr . A recent series of studies also employed the PheRS editing domain inserted into a TyrRS variant; however, in this case the goal was to change the enantioselectivity of TyrRS from L -Tyr-tRNA Tyr to D -Tyr-tRNA Tyr [71, 72], resulting in an approximately 2.6-fold shift in the enantioselectivity of the enzyme towards the formation of D -Tyr-tRNA Tyr [71, 72]. …”