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PMID: 25385624 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S.

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 111 ·No. 47 ·2014-11-25 ·Pages 16724-9

Guo LT, Wang YS, Nakamura A, Eiler D, Kavran JM, Wong M, Kiessling LL, Steitz TA, O'Donoghue P, Söll D

Abstract

Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA(Pyl) have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate N(ε)-acetyl-Lys (AcK) onto tRNA(Pyl). Here, we examine an N(ε)-acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids.

Keywords
aminoacyl-tRNA synthetase genetic code genetic selection posttranslational modification synthetic biology
MeSH Terms
Amino Acyl-tRNA Synthetases/metabolism Directed Molecular Evolution Kinetics Lysine/metabolism
Chemicals
Amino Acyl-tRNA Synthetases Lysine
Authors & Affiliations
10 authors, click to expand affiliations / ORCID
Guo Li-Tao
Departments of Molecular Biophysics & Biochemistry and.
Wang Yane-Shih
Departments of Molecular Biophysics & Biochemistry and.
Nakamura Akiyoshi
Departments of Molecular Biophysics & Biochemistry and Chemistry, and.
Eiler Daniel
Departments of Molecular Biophysics & Biochemistry and.
Kavran Jennifer M
Departments of Molecular Biophysics & Biochemistry and.
Wong Margaret
Novartis Institutes of Biomedical Research, Cambridge, MA 02139;
Kiessling Laura L
Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706; and.
Steitz Thomas A
Departments of Molecular Biophysics & Biochemistry and Chemistry, and Howard Hughes Medical Institute, Yale University, New Haven, CT 06510;
O'Donoghue Patrick
Departments of Biochemistry and Chemistry, The University of Western Ontario, London, ON, Canada N6A 5C1 [email protected] [email protected].
Söll Dieter
Departments of Molecular Biophysics & Biochemistry and Chemistry, and [email protected] [email protected].
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
1091-6490
DOI
10.1073/pnas.1419737111
Email
[email protected]
[email protected]
Published
2014-11-25
Epub
2014-00-10
Pages
16724-9
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC4250173
Subset
IM
Grants
NIGMS NIH HHS · R37 GM022854 · United States
NIGMS NIH HHS · R01 GM055984 · United States
NIGMS NIH HHS · R01 GM022854 · United States
NIGMS NIH HHS · P41 GM111244 · United States
Howard Hughes Medical Institute · United States
NIGMS NIH HHS · P01 GM022778 · United States
NIGMS NIH HHS · GM22854 · United States
NIGMS NIH HHS · GM055984 · United States
Databases
PDB
4Q6G, 4TQD, 4TQF
External Links
PubMed source DOI: 10.1073/pnas.1419737111 PMC Full Text View journal details
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