Computational design and experimental characterization of a photo-controlled mRNA-cap guanine-N7 methyltransferase.

Autor:	Reichert D; Institute of Biochemistry, University of Münster, Correnstraße 36 Münster 48149 Germany a.rentmeister@uni-muenster.de.; Cells in Motion Interfaculty Center, University of Münster Münster 48149 Germany., Schepers H; Institute of Biochemistry, University of Münster, Correnstraße 36 Münster 48149 Germany a.rentmeister@uni-muenster.de., Simke J; Organic Chemistry Institute, University of Münster, Corrensstraße 36 Münster 48149 Germany., Lechner H; Department for Biochemistry, University of Bayreuth, Universitätsstraße 30 Bayreuth 95447 Germany.; Institute of Biochemistry, Graz University of Technology, Petersgasse 10-12/II Graz 8010 Austria., Dörner W; Institute of Biochemistry, University of Münster, Correnstraße 36 Münster 48149 Germany a.rentmeister@uni-muenster.de., Höcker B; Department for Biochemistry, University of Bayreuth, Universitätsstraße 30 Bayreuth 95447 Germany., Ravoo BJ; Cells in Motion Interfaculty Center, University of Münster Münster 48149 Germany.; Organic Chemistry Institute, University of Münster, Corrensstraße 36 Münster 48149 Germany., Rentmeister A; Institute of Biochemistry, University of Münster, Correnstraße 36 Münster 48149 Germany a.rentmeister@uni-muenster.de.; Cells in Motion Interfaculty Center, University of Münster Münster 48149 Germany.
Jazyk:	angličtina
Zdroj:	RSC chemical biology [RSC Chem Biol] 2021 Jun 29; Vol. 2 (5), pp. 1484-1490. Date of Electronic Publication: 2021 Jun 29 (Print Publication: 2021).
DOI:	10.1039/d1cb00109d
Abstrakt:	The spatial and temporal control of gene expression at the post-transcriptional level is essential in eukaryotic cells and developing multicellular organisms. In recent years optochemical and optogenetic tools have enabled the manipulation and investigation of many steps in the involved processes. However, examples for light-mediated control of eukaryotic mRNA processing and the responsible enzymes are still rare. In particular, methylation of the 5' cap of mRNA is required for ribosome assembly, and the responsible guanine-N7 methyltransferase (MTase) from E. cuniculi (Ecm1) proved suitable for activating translation. Here, we report on a photoswitchable MTase obtained by bridging the substrate-binding cleft of Ecm1 with a tetra- ortho -methoxy-azobenzene. This azobenzene derivative is characterized by efficient trans -to- cis isomerization using red light at 615 nm. Starting from a cysteine-free Ecm1 variant (ΔCys), we used a computational approach to identify suitable conjugation sites for the azobenzene moiety. We created and characterized the four best-ranked variants, each featuring two appropriately positioned cysteines close to the substrate-binding cleft. Conjugating and crosslinking the azobenzene between C149/C155 in a designed Ecm1 variant (VAR3-Az) enabled light-dependent modulation of the MTase activity and showed a 50% higher activity for the cis form than the trans -form of the azobenzene conjugated to VAR3-Az. Competing Interests: The authors declare no conflict of interest. (This journal is © The Royal Society of Chemistry.)
Databáze:	MEDLINE
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