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

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Diphthamide is a post-translational modification of elongation factor 2 (EF2) crucial for accurate mRNA translation and protein synthesis in eukaryotes.
• The Dph1•Dph2 dimer is a non-canonical radical SAM enzyme responsible for diphthamide synthesis, utilizing iron-sulfur (FeS) clusters.

Purpose of the Study:

• To identify and characterize the S-adenosyl-methionine (SAM) binding pocket in the Dph1•Dph2 enzyme from *Saccharomyces cerevisiae*.
• To elucidate the role of this SAM-binding pocket in the synthesis of diphthamide and its importance for EF2 function.

Main Methods:

• Bioinformatic analysis to predict SAM-binding pocket based on archaeal orthologues.
• Site-directed mutagenesis of the *DPH1* gene.
• Functional characterization using assay diagnostics for diphthamide synthesis and EF2 activity.

Main Results:

• A conserved SAM-binding pocket was predicted in Dph1, located near the FeS cluster domain.
• Mutagenesis of residues within this SAM pocket abolished diphthamide synthesis in vivo, confirming its essentiality.
• Structural modeling suggested specific residues critical for SAM cleavage and 3-amino-3-carboxy-propyl (ACP) radical formation.

Conclusions:

• The identified SAM-binding pocket in Dph1 is essential for the synthesis of diphthamide, a modification vital for protein synthesis.
• Dph1•Dph2 employs a distinct mechanism for SAM cleavage compared to classical radical SAM enzymes, generating an ACP radical instead of a dAdo radical.