YBR246W is required for the third step of diphthamide biosynthesis

Abstract

Diphthamide, the target of diphtheria toxin, is a post-translationally modified histidine residue that is found in archaeal and eukaryotic translation elongation factor 2. The biosynthesis and function of this modification has attracted the interest of many biochemists for decades. The biosynthesis has been known to proceed in three steps. Proteins required for the first and second steps have been identified, but the protein(s) required for the last step have remained elusive. Here we demonstrate that the YBR246W gene in yeast is required for the last step of diphthamide biosynthesis, as the deletion of YBR246W leads to the accumulation of diphthine, which is the enzymatic product of the second step of the biosynthesis. This discovery will provide important information leading to the complete elucidation of the full biosynthesis pathway of diphthamide.

Figure 1

In vitroADP-ribosylation using Rh-NAD. The upper panel was Coomassie blue-stained gel showing the eEF-2 proteins and the lower panel showed the corresponding fluorescence labeling. The source strains from which the eEF-2 proteins were purified were labeled above. No DT was added in lane 1–3 and 100 nM DT was added to lane 4–6. Reactions were carried out at 30°C for 20 min.

Figure 2

DT sensitivity assays of WT and deletion strains. (A) The strains were transformed with pLMY101, which encodes the catalytic fragment of DT, and were grown on 2% Gal medium. (B) WT, Δdph2 and Δybr246w were grown on 2% Raf plus varying concentrations of Gal. The growth on 2% Glc was shown as a control.

Figure 3

In vitroADP-ribosylation assay with two different concentrations of DT. The upper panel was the Coomassie blue-stained gel showing the eEF-2 proteins and the lower panel showed the corresponding fluorescence labeling. The source strains from which the eEF-2 proteins were purified were labeled above. Reactions shown in lane 1–3 contained 0.1 μM of DT and those shown in lane 4–6 contained 10 μM of DT. Reactions were carried out at 30°C for 60 min.

Figure 4

Extracted ion chromatograms of diphthamide and diphthine from different strains. The peaks corresponding to diphthamide and diphthine containing peptides were highlighted in grey. The peptides carried 4 positive charges. The retention time (RT) and peak area integration (MA) were shown above the highlighted peaks.

Figure 5

MS/MS spectra of peptides containing diphthamide (A) and diphthine (B). A neutral loss of the trimethyl amino group was observed in both spectra.

Scheme 1

Biosynthesis pathway of diphthamide.