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Main Title: Computational Aminoacyl-tRNA Synthetase Library Design for Photocaged Tyrosine
Author(s): Baumann, Tobias
Hauf, Matthias
Richter, Florian
Albers, Suki
Möglich, Andreas
Ignatova, Zoya
Budisa, Nediljko
Type: Article
Language Code: en
Abstract: Engineering aminoacyl-tRNA synthetases (aaRSs) provides access to the ribosomal incorporation of noncanonical amino acids via genetic code expansion. Conventional targeted mutagenesis libraries with 5–7 positions randomized cover only marginal fractions of the vast sequence space formed by up to 30 active site residues. This frequently results in selection of weakly active enzymes. To overcome this limitation, we use computational enzyme design to generate a focused library of aaRS variants. For aaRS enzyme redesign, photocaged ortho-nitrobenzyl tyrosine (ONBY) was chosen as substrate due to commercial availability and its diverse applications. Diversifying 17 first- and second-shell sites and performing conventional aaRS positive and negative selection resulted in a high-activity aaRS. This MjTyrRS variant carries ten mutations and outperforms previously reported ONBY-specific aaRS variants isolated from traditional libraries. In response to a single in-frame amber stop codon, it mediates the in vivo incorporation of ONBY with an efficiency matching that of the wild type MjTyrRS enzyme acylating cognate tyrosine. These results exemplify an improved general strategy for aaRS library design and engineering.
Issue Date: 11-May-2019
Date Available: 14-May-2019
DDC Class: 570 Biowissenschaften; Biologie
540 Chemie und zugeordnete Wissenschaften
Subject(s): enzyme design
noncanonical amino acids
protein modification
directed evolution
gene libraries
genetic code expansion
unnatural amino acids
protein engineering
Sponsor/Funder: DFG, 414044773, Open Access Publizieren 2019 - 2020 / Technische Universität Berlin
DFG, 207100805, FOR 1805: Einfluss der Ribosomendynamik auf Regulation der Geschwindigkeit und Genauigkeit der Translation
DFG, 390540038, EXC 2008: Vereinigung von Systemen in der Katalyse
Journal Title: International Journal of Molecular Sciences
Publisher: MDPI
Publisher Place: Basel
Volume: 20
Issue: 9
Article Number: 2343
Publisher DOI: 10.3390/ijms20092343
EISSN: 1422-0067
Appears in Collections:FG Biokatalyse » Publications

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