Functional Integrity of Radical SAM Enzyme Dph1•Dph2 Requires Non-Canonical Cofactor Motifs with Tandem Cysteines

• Published in: Biomolecules (Basel, Switzerland) Vol. 14; no. 4; p. 470
• Main Authors: Ütkür, Koray, Mayer, Klaus, Liu, Shihui, Brinkmann, Ulrich, Schaffrath, Raffael
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 01.04.2024
• Subjects: Amino Acid Motifs; Analysis; Antibodies; Carbon-Sulfur Lyases - chemistry; Carbon-Sulfur Lyases - genetics; Carbon-Sulfur Lyases - metabolism; Cycloheximide; Cysteine; Cysteine - chemistry; Cysteine - genetics; Cysteine - metabolism; cysteine ligands; diphthamide modification; Dph1•Dph2; eEF2; Enzymes; Histidine - analogs & derivatives; Identification and classification; iron sulfur cluster; Kinases; Ligands; Methionine; Mutagenesis; Mutagenesis, Site-Directed; Protein Multimerization; Proteins; radical SAM enzyme; Repressor Proteins; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Toxins; Yeast; Germany; United States > US; Basel Switzerland; Switzerland; iron sulfur cluster; eEF2; diphthamide modification; Dph1•Dph2; cysteine ligands; radical SAM enzyme; Saccharomyces cerevisiae; ADP-ribosylation
• ISSN: 2218-273X; 2218-273X
• DOI: 10.3390/biom14040470

The Dph1•Dph2 heterodimer from yeast is a radical SAM (RS) enzyme that generates the 3-amino-3-carboxy-propyl (ACP) precursor for diphthamide, a clinically relevant modification on eukaryotic elongation factor 2 (eEF2). ACP formation requires SAM cleavage and atypical Cys-bound Fe-S clusters in each Dph1 and Dph2 subunit. Intriguingly, the first Cys residue in each motif is found next to another ill-defined cysteine that we show is conserved across eukaryotes. As judged from structural modeling, the orientation of these tandem cysteine motifs (TCMs) suggests a candidate Fe-S cluster ligand role. Hence, we generated, by site-directed and mutagenesis, Dph1•Dph2 variants with cysteines from each TCM replaced individually or in combination by serines. Assays diagnostic for diphthamide formation in vivo reveal that while single substitutions in the TCM of Dph2 cause mild defects, double mutations almost entirely inactivate the RS enzyme. Based on enhanced Dph1 and Dph2 subunit instability in response to cycloheximide chases, the variants with Cys substitutions in their cofactor motifs are particularly prone to protein degradation. In sum, we identify a fourth functionally cooperative Cys residue within the Fe-S motif of Dph2 and show that the Cys-based cofactor binding motifs in Dph1 and Dph2 are critical for the structural integrity of the dimeric RS enzyme in vivo.