title: An Ancestral Tryptophanyl-tRNA Synthetase Precursor Achieves High Catalytic Rate Enhancement without Ordered Ground-State Tertiary Structures.
abstract: Urzymes-short, active core modules derived from enzyme superfamilies-prepared from the two aminoacyl-tRNA synthetase (aaRS) classes contain only the modules shared by all related family members. They have been described as models for ancestral forms. Understanding them currently depends on inferences drawn from the crystal structures of the full-length enzymes. As aaRS Urzymes lack much of the mass of modern aaRS's, retaining only a small portion of the hydrophobic cores of the full-length enzymes, it is desirable to characterize their structures. We report preliminary characterization of (15)N tryptophanyl-tRNA synthetase Urzyme by heteronuclear single quantum coherence (HSQC) NMR spectroscopy supplemented by circular dichroism, thermal melting, and induced fluorescence of bound dye. The limited dispersion of (1)H chemical shifts (0.5 ppm) is inconsistent with a narrow ensemble of well-packed structures in either free or substrate-bound forms, although the number of resonances from the bound state increases, indicating a modest, ligand-dependent gain in structure. Circular dichroism spectroscopy shows the presence of helices and evidence of cold denaturation, and all ligation states induce Sypro Orange fluorescence at ambient temperatures. Although the term "molten globule" is difficult to define precisely, these characteristics are consistent with most such definitions. Active-site titration shows that a majority of molecules retain ∼60% of the transition state stabilization free energy observed in modern synthetases. In contrast to the conventional view that enzymes require stable tertiary structures, we conclude that a highly flexible ground-state ensemble can nevertheless bind tightly to the transition state for amino acid activation.
authors.affiliation: Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, ‡Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 25799, United States.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Sapienza
authors.affiliation: Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, ‡Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 25799, United States.
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.affiliation: Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, ‡Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 25799, United States.
authors.forename: Tishan
authors.initials: T
authors.lastname: Williams
authors.affiliation: Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, ‡Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 25799, United States.
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Lee
authors.affiliation: Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, ‡Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 25799, United States.
authors.forename: Charles W
authors.initials: CW
authors.lastname: Carter
mesh_terms: D055162:Biocatalysis; D002942:Circular Dichroism; D004792:Enzyme Precursors / Q000737:chemistry* / Q000302:isolation & purification; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D057927:Hydrophobic and Hydrophilic Interactions; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D066244:Proton Magnetic Resonance Spectroscopy; D014369:Tryptophan-tRNA Ligase / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D004792:Enzyme Precursors; D014369:Tryptophan-tRNA Ligase