title: Aminoacyl-tRNA substrate and enzyme backbone atoms contribute to translational quality control by YbaK.
abstract: Amino acids are covalently attached to their corresponding transfer RNAs (tRNAs) by aminoacyl-tRNA synthetases. Proofreading mechanisms exist to ensure that high fidelity is maintained in this key step in protein synthesis. Prolyl-tRNA synthetase (ProRS) can misacylate cognate tRNA(Pro) with Ala and Cys. The cis-editing domain of ProRS (INS) hydrolyzes Ala-tRNA(Pro), whereas Cys-tRNA(Pro) is hydrolyzed by a single domain editing protein, YbaK, in trans. Previous studies have proposed a model of substrate-binding by bacterial YbaK and elucidated a substrate-assisted mechanism of catalysis. However, the microscopic steps in this mechanism have not been investigated. In this work, we carried out biochemical experiments together with a detailed hybrid quantum mechanics/molecular mechanics study to investigate the mechanism of catalysis by Escherichia coli YbaK. The results support a mechanism wherein cyclization of the substrate Cys results in cleavage of the Cys-tRNA ester bond. Protein side chains do not play a significant role in YbaK catalysis. Instead, protein backbone atoms play crucial roles in stabilizing the transition state, while the product is stabilized by the 2'-OH of the tRNA.
authors.affiliation: Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA.
authors.forename: Sandeep
authors.initials: S
authors.lastname: Kumar
authors.forename: Mom
authors.initials: M
authors.lastname: Das
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Hadad
authors.forename: Karin
authors.initials: K
authors.lastname: Musier-Forsyth
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D055162:Biocatalysis; D002352:Carrier Proteins / Q000378:metabolism*; D020134:Catalytic Domain; D003500:Cyclization; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006868:Hydrolysis; D007536:Isomerism; D008958:Models, Molecular; D014176:Protein Biosynthesis; D011789:Quantum Theory; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D012346:RNA, Transfer, Amino Acyl; C584682:YbaK protein, E coli; D000604:Amino Acyl-tRNA Synthetases; D003545:Cysteine