title: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center in Shreveport , 1501 Kings Highway, Shreveport, Louisiana 71130, United States.
authors.forename: Charles J
authors.initials: CJ
authors.lastname: Richardson
authors.affiliation: Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center in Shreveport , 1501 Kings Highway, Shreveport, Louisiana 71130, United States.
authors.forename: Eric A
authors.initials: EA
authors.lastname: First
mesh_terms: D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D043902:Pyrococcus horikoshii / Q000201:enzymology*; D013237:Stereoisomerism; D014448:Tyrosine-tRNA Ligase / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D014448:Tyrosine-tRNA Ligase