Multistep Engineering of Pyrrolysyl-tRNA Synthetase to Genetically Encode Nε-(o-Azidobenzyloxycarbonyl) lysine for Site-Specific Protein Modification

Tatsuo Yanagisawa; Ryohei Ishii; Ryuya Fukunaga; Takatsugu Kobayashi; Kensaku Sakamoto; Shigeyuki Yokoyama

doi:10.1016/j.chembiol.2008.10.004

Multistep Engineering of Pyrrolysyl-tRNA Synthetase to Genetically Encode N^ε-(o-Azidobenzyloxycarbonyl) lysine for Site-Specific Protein Modification

Tatsuo Yanagisawa
, Ryohei Ishii
, Ryuya Fukunaga
, Takatsugu Kobayashi
, Kensaku Sakamoto
, Shigeyuki Yokoyama

Research output: Contribution to journal › Article › peer-review

211 Scopus citations

Abstract

Pyrrolysyl-tRNA synthetase (PylRS) esterifies pyrrolysine to tRNA^Pyl. In this study, N^ε-(tert-butyloxycarbonyl)-L-lysine (BocLys) and N^ε-allyloxycarbonyl-L-lysine (AlocLys) were esterified to tRNA^Pyl by PylRS. Crystal structures of a PylRS catalytic fragment complexed with BocLys and an ATP analog and with AlocLys-AMP revealed that PylRS requires an N^ε-carbonyl group bearing a substituent with a certain size. A PylRS(Y384F) mutant obtained by random screening exhibited higher in vitro aminoacylation and in vivo amber suppression activities with BocLys, AlocLys, and pyrrolysine than those of the wild-type PylRS. Furthermore, the structure-based Y306A mutation of PylRS drastically increased the in vitro aminoacylation activity for N^ε-benzyloxycarbonyl-L-lysine (ZLys). A PylRS with both the Y306A and Y384F mutations enabled the large-scale preparation (>10 mg per liter medium) of proteins site-specifically containing N^ε-(o-azidobenzyloxycarbonyl)-L-lysine (AzZLys). The AzZLys-containing protein was labeled with a fluorescent probe, by Staudinger ligation.

Original language	English (US)
Pages (from-to)	1187-1197
Number of pages	11
Journal	Chemistry and Biology
Volume	15
Issue number	11
DOIs	https://doi.org/10.1016/j.chembiol.2008.10.004
State	Published - Nov 24 2008
Externally published	Yes

Keywords

CHEMBIO
DNA
PROTEINS

ASJC Scopus subject areas

Biochemistry
Molecular Medicine
Molecular Biology
Pharmacology
Drug Discovery
Clinical Biochemistry

Access to Document

10.1016/j.chembiol.2008.10.004

Cite this

@article{e383e3ff23304931bf7f7e11e2500421,

title = "Multistep Engineering of Pyrrolysyl-tRNA Synthetase to Genetically Encode Nε-(o-Azidobenzyloxycarbonyl) lysine for Site-Specific Protein Modification",

abstract = "Pyrrolysyl-tRNA synthetase (PylRS) esterifies pyrrolysine to tRNAPyl. In this study, Nε-(tert-butyloxycarbonyl)-L-lysine (BocLys) and Nε-allyloxycarbonyl-L-lysine (AlocLys) were esterified to tRNAPyl by PylRS. Crystal structures of a PylRS catalytic fragment complexed with BocLys and an ATP analog and with AlocLys-AMP revealed that PylRS requires an Nε-carbonyl group bearing a substituent with a certain size. A PylRS(Y384F) mutant obtained by random screening exhibited higher in vitro aminoacylation and in vivo amber suppression activities with BocLys, AlocLys, and pyrrolysine than those of the wild-type PylRS. Furthermore, the structure-based Y306A mutation of PylRS drastically increased the in vitro aminoacylation activity for Nε-benzyloxycarbonyl-L-lysine (ZLys). A PylRS with both the Y306A and Y384F mutations enabled the large-scale preparation (>10 mg per liter medium) of proteins site-specifically containing Nε-(o-azidobenzyloxycarbonyl)-L-lysine (AzZLys). The AzZLys-containing protein was labeled with a fluorescent probe, by Staudinger ligation.",

keywords = "CHEMBIO, DNA, PROTEINS",

author = "Tatsuo Yanagisawa and Ryohei Ishii and Ryuya Fukunaga and Takatsugu Kobayashi and Kensaku Sakamoto and Shigeyuki Yokoyama",

note = "Funding Information: We would like to thank the staff of the beamline BL41XU at SPring-8 (Harima, Japan), as well as the staff of the BL5A and AR-NW12 beamlines at the Photon Factory (Tsukuba, Japan). We also thank Shun-ichi Sekine, Takuhiro Ito (The University of Tokyo), and Toru Sengoku (RIKEN) for assisting with the data collection, as well as for helpful discussions. We are grateful to Ko-ichiro Kodama (The University of Tokyo) for helpful discussions about the chemistry of non-natural amino acids. We would like to thank Yutaka Muto (RIKEN) for help with the NMR measurement. We thank Miwako Asanuma, Ryogo Akasaka, Machiko Yamaguchi-Hirafuji, Nobuko Maoka, Takahito Mukai, Takaho Terada, Mikako Shirouzu, and Hiroshi Hirota (RIKEN) for mass spectrometry analyses. We would also like to thank Tomomi Sumida for technical assistance, and Azusa Ishii, Kiyomi Yajima, and Tomoko Nakayama for clerical assistance. This work was supported in part by Grants-in-Aid for Scientific Research in Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, the Project for the Development of Fundamental Technology for Protein Analyses, the Targeted Proteins Research Program (TPRP), and the RIKEN Structural Genomics/Proteomics Initiative (RSGI), in the National Project on Protein Structural and Functional Analyses, MEXT. T.K. was supported by the special postdoctoral researcher program of RIKEN. T.Y. expresses his deepest gratitude to the late Emiko Fusatomi for her continuous encouragement and the peace of mind she gave him. ",

year = "2008",

month = nov,

day = "24",

doi = "10.1016/j.chembiol.2008.10.004",

language = "English (US)",

volume = "15",

pages = "1187--1197",

journal = "Chemistry and Biology",

issn = "1074-5521",

publisher = "Elsevier Limited",

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TY - JOUR

T1 - Multistep Engineering of Pyrrolysyl-tRNA Synthetase to Genetically Encode Nε-(o-Azidobenzyloxycarbonyl) lysine for Site-Specific Protein Modification

AU - Yanagisawa, Tatsuo

AU - Ishii, Ryohei

AU - Fukunaga, Ryuya

AU - Kobayashi, Takatsugu

AU - Sakamoto, Kensaku

AU - Yokoyama, Shigeyuki

N1 - Funding Information: We would like to thank the staff of the beamline BL41XU at SPring-8 (Harima, Japan), as well as the staff of the BL5A and AR-NW12 beamlines at the Photon Factory (Tsukuba, Japan). We also thank Shun-ichi Sekine, Takuhiro Ito (The University of Tokyo), and Toru Sengoku (RIKEN) for assisting with the data collection, as well as for helpful discussions. We are grateful to Ko-ichiro Kodama (The University of Tokyo) for helpful discussions about the chemistry of non-natural amino acids. We would like to thank Yutaka Muto (RIKEN) for help with the NMR measurement. We thank Miwako Asanuma, Ryogo Akasaka, Machiko Yamaguchi-Hirafuji, Nobuko Maoka, Takahito Mukai, Takaho Terada, Mikako Shirouzu, and Hiroshi Hirota (RIKEN) for mass spectrometry analyses. We would also like to thank Tomomi Sumida for technical assistance, and Azusa Ishii, Kiyomi Yajima, and Tomoko Nakayama for clerical assistance. This work was supported in part by Grants-in-Aid for Scientific Research in Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, the Project for the Development of Fundamental Technology for Protein Analyses, the Targeted Proteins Research Program (TPRP), and the RIKEN Structural Genomics/Proteomics Initiative (RSGI), in the National Project on Protein Structural and Functional Analyses, MEXT. T.K. was supported by the special postdoctoral researcher program of RIKEN. T.Y. expresses his deepest gratitude to the late Emiko Fusatomi for her continuous encouragement and the peace of mind she gave him.

PY - 2008/11/24

Y1 - 2008/11/24

N2 - Pyrrolysyl-tRNA synthetase (PylRS) esterifies pyrrolysine to tRNAPyl. In this study, Nε-(tert-butyloxycarbonyl)-L-lysine (BocLys) and Nε-allyloxycarbonyl-L-lysine (AlocLys) were esterified to tRNAPyl by PylRS. Crystal structures of a PylRS catalytic fragment complexed with BocLys and an ATP analog and with AlocLys-AMP revealed that PylRS requires an Nε-carbonyl group bearing a substituent with a certain size. A PylRS(Y384F) mutant obtained by random screening exhibited higher in vitro aminoacylation and in vivo amber suppression activities with BocLys, AlocLys, and pyrrolysine than those of the wild-type PylRS. Furthermore, the structure-based Y306A mutation of PylRS drastically increased the in vitro aminoacylation activity for Nε-benzyloxycarbonyl-L-lysine (ZLys). A PylRS with both the Y306A and Y384F mutations enabled the large-scale preparation (>10 mg per liter medium) of proteins site-specifically containing Nε-(o-azidobenzyloxycarbonyl)-L-lysine (AzZLys). The AzZLys-containing protein was labeled with a fluorescent probe, by Staudinger ligation.

AB - Pyrrolysyl-tRNA synthetase (PylRS) esterifies pyrrolysine to tRNAPyl. In this study, Nε-(tert-butyloxycarbonyl)-L-lysine (BocLys) and Nε-allyloxycarbonyl-L-lysine (AlocLys) were esterified to tRNAPyl by PylRS. Crystal structures of a PylRS catalytic fragment complexed with BocLys and an ATP analog and with AlocLys-AMP revealed that PylRS requires an Nε-carbonyl group bearing a substituent with a certain size. A PylRS(Y384F) mutant obtained by random screening exhibited higher in vitro aminoacylation and in vivo amber suppression activities with BocLys, AlocLys, and pyrrolysine than those of the wild-type PylRS. Furthermore, the structure-based Y306A mutation of PylRS drastically increased the in vitro aminoacylation activity for Nε-benzyloxycarbonyl-L-lysine (ZLys). A PylRS with both the Y306A and Y384F mutations enabled the large-scale preparation (>10 mg per liter medium) of proteins site-specifically containing Nε-(o-azidobenzyloxycarbonyl)-L-lysine (AzZLys). The AzZLys-containing protein was labeled with a fluorescent probe, by Staudinger ligation.

KW - CHEMBIO

KW - DNA

KW - PROTEINS

UR - https://www.scopus.com/pages/publications/56049106840

UR - https://www.scopus.com/pages/publications/56049106840#tab=citedBy

U2 - 10.1016/j.chembiol.2008.10.004

DO - 10.1016/j.chembiol.2008.10.004

M3 - Article

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AN - SCOPUS:56049106840

SN - 1074-5521

VL - 15

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EP - 1197

JO - Chemistry and Biology

JF - Chemistry and Biology

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