DPH1 Gene Mutations Identify a Candidate SAM Pocket in Radical Enzyme Dph1•Dph2 for Diphthamide Synthesis on EF2

• Published in: Biomolecules (Basel, Switzerland) Vol. 13; no. 11; p. 1655
• Main Authors: Ütkür, Koray, Schmidt, Sarina, Mayer, Klaus, Klassen, Roland, Brinkmann, Ulrich, Schaffrath, Raffael
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 16.11.2023
• Subjects: Amino acids; Antibodies; Diphtheria; diphtheria toxin; Dph1•Dph2; EF2 diphthamide modification; Enzymes; Histidine; Histidine - chemistry; Humans; Iron sulfides; Kinases; Methionine; Minor Histocompatibility Antigens; mRNA; Mutagenesis; Mutation; Peptide Elongation Factor 2 - metabolism; Protein biosynthesis; Proteins; radical SAM enzymes; S-Adenosylmethionine - metabolism; Saccharomyces cerevisiae; Saccharomyces cerevisiae - metabolism; SAM; Transcription; Tumor Suppressor Proteins - metabolism; Yeast; United States > US; ADP ribosylation; EF2 diphthamide modification; Dph1•Dph2; diphtheria toxin; radical SAM enzymes; Saccharomyces cerevisiae; SAM
• ISSN: 2218-273X; 2218-273X
• DOI: 10.3390/biom13111655

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 . We predict an SAM-binding pocket near the FeS cluster domain that is conserved across eukaryotes in Dph1 but not Dph2. Site-directed 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.