Datum
2023-11-16Schlagwort
570 Biowissenschaften, Biologie Saccharomyces cerevisiaeRadikal <Chemie>EnzymDiphtherieADP-RibosylierungMetadata
Zur Langanzeige
Aufsatz
DPH1 Gene Mutations Identify a Candidate SAM Pocket in Radical Enzyme Dph1 Dph2 for Diphthamide Synthesis on EF2
Zusammenfassung
In eukaryotes, the Dph1•Dph2 dimer is a non-canonical radical SAM enzyme. Using iron-sulfur (FeS) clusters, it cleaves the cosubstrate S-adenosyl-methionine (SAM) to form a 3-amino-3-carboxy-propyl (ACP) radical for the synthesis of diphthamide. The latter decorates a histidine residue on elongation factor 2 (EF2) conserved from archaea to yeast and humans and is important for accurate mRNA translation and protein synthesis. Guided by evidence from archaeal orthologues, we searched for a putative SAM-binding pocket in Dph1•Dph2 from Saccharomyces cerevisiae. We predict an SAM-binding pocket near the FeS cluster domain that is conserved across eukaryotes in Dph1 but not Dph2. Site-directed DPH1 mutagenesis and functional characterization through assay diagnostics for the loss of diphthamide reveal that the SAM pocket is essential for synthesis of the décor on EF2 in vivo. Further evidence from structural modeling suggests particularly critical residues close to the methionine moiety of SAM. Presumably, they facilitate a geometry specific for SAM cleavage and ACP radical formation that distinguishes Dph1•Dph2 from classical radical SAM enzymes, which generate canonical 5′-deoxyadenosyl (dAdo) radicals.
Zitierform
In: Biomolecules Volume 13 / Issue 11 (2023-11-16) eissn:2218-273XFörderhinweis
Gefördert durch den Publikationsfonds der Universität KasselZitieren
@article{doi:10.17170/kobra-202312089194,
author={Schaffrath, Raffael and Ütkür, Koray and Mayer, Klaus and Schmidt, Sarina and Klassen, Roland and Brinkmann, Ulrich},
title={DPH1 Gene Mutations Identify a Candidate SAM Pocket in Radical Enzyme Dph1 Dph2 for Diphthamide Synthesis on EF2},
journal={Biomolecules},
year={2023}
}
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2023-12-08T15:17:35Z 2023-12-08T15:17:35Z 2023-11-16 doi:10.17170/kobra-202312089194 http://hdl.handle.net/123456789/15279 Gefördert durch den Publikationsfonds der Universität Kassel eng Namensnennung 4.0 International http://creativecommons.org/licenses/by/4.0/ Saccharomyces cerevisiae SAM radical SAM enzymes EF2 diphthamide modification Dph1•Dph2 diphtheria toxin ADP ribosylation 570 DPH1 Gene Mutations Identify a Candidate SAM Pocket in Radical Enzyme Dph1 Dph2 for Diphthamide Synthesis on EF2 Aufsatz In eukaryotes, the Dph1•Dph2 dimer is a non-canonical radical SAM enzyme. Using iron-sulfur (FeS) clusters, it cleaves the cosubstrate S-adenosyl-methionine (SAM) to form a 3-amino-3-carboxy-propyl (ACP) radical for the synthesis of diphthamide. The latter decorates a histidine residue on elongation factor 2 (EF2) conserved from archaea to yeast and humans and is important for accurate mRNA translation and protein synthesis. Guided by evidence from archaeal orthologues, we searched for a putative SAM-binding pocket in Dph1•Dph2 from Saccharomyces cerevisiae. We predict an SAM-binding pocket near the FeS cluster domain that is conserved across eukaryotes in Dph1 but not Dph2. Site-directed DPH1 mutagenesis and functional characterization through assay diagnostics for the loss of diphthamide reveal that the SAM pocket is essential for synthesis of the décor on EF2 in vivo. Further evidence from structural modeling suggests particularly critical residues close to the methionine moiety of SAM. Presumably, they facilitate a geometry specific for SAM cleavage and ACP radical formation that distinguishes Dph1•Dph2 from classical radical SAM enzymes, which generate canonical 5′-deoxyadenosyl (dAdo) radicals. open access Schaffrath, Raffael Ütkür, Koray Mayer, Klaus Schmidt, Sarina Klassen, Roland Brinkmann, Ulrich 13 Seiten doi:10.3390/biom13111655 Saccharomyces cerevisiae Radikal <Chemie> Enzym Diphtherie ADP-Ribosylierung publishedVersion eissn:2218-273X Issue 11 Biomolecules Volume 13 false 1655
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