The asymmetric function of Dph1-Dph2 heterodimer in diphthamide biosynthesis

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Diphthamide is a unique post-translational modification of translation elongation factor 2 (EF2).
• This modification is crucial in eukaryotes and archaea, targeting EF2 for diphtheria toxin.
• Radical SAM enzymes, like Dph1-Dph2, are involved in the initial step of diphthamide biosynthesis.

Purpose of the Study:

• To investigate the in vivo requirement of [4Fe-4S] cluster-binding cysteine residues in the eukaryotic Dph1-Dph2 heterodimer.
• To elucidate the functional roles of the individual [4Fe-4S] clusters within the Dph1-Dph2 complex during diphthamide biosynthesis.

Main Methods:

• Site-directed mutagenesis of [4Fe-4S] cluster-binding cysteine residues in Dph1 and Dph2.
• In vivo assays to assess diphthamide biosynthesis in mutant strains.
• In vitro reconstitution experiments using purified Dph1-Dph2 mutants and the Dph3/Cbr1/NADH reducing system.

Main Results:

• The [4Fe-4S] cluster-binding cysteine residues in both Dph1 and Dph2 subunits are essential for diphthamide biosynthesis in vivo.
• In vitro experiments indicated that the Dph1 [4Fe-4S] cluster plays a direct catalytic role.
• The Dph2 [4Fe-4S] cluster was found to facilitate the reduction of the Dph1 cluster by the cellular reducing machinery.

Conclusions:

• The eukaryotic Dph1-Dph2 heterodimer exhibits asymmetric functional roles for its [4Fe-4S] clusters.
• This study provides insights into the mechanism of iron-sulfur cluster reduction in radical SAM enzymes.
• Understanding these mechanisms is vital for comprehending essential cellular processes and potential therapeutic targets.