Dph3 is an electron donor for Dph1-Dph2 in the first step of eukaryotic diphthamide biosynthesis

Abstract

Diphthamide, the target of diphtheria toxin, is a unique posttranslational modification on translation elongation factor 2 (EF2) in archaea and eukaryotes. The biosynthesis of diphthamide was proposed to involve three steps. The first step is the transfer of the 3-amino-3-carboxypropyl group from S-adenosyl-l-methionine (SAM) to the histidine residue of EF2, forming a C-C bond. Previous genetic studies showed this step requires four proteins in eukaryotes, Dph1-Dph4. However, the exact molecular functions for the four proteins are unknown. Previous study showed that Pyrococcus horikoshii Dph2 (PhDph2), a novel iron-sulfur cluster-containing enzyme, forms a homodimer and is sufficient for the first step of diphthamide biosynthesis in vitro. Here we demonstrate by in vitro reconstitution that yeast Dph1 and Dph2 form a complex (Dph1-Dph2) that is equivalent to the homodimer of PhDph2 and is sufficient to catalyze the first step in vitro in the presence of dithionite as the reductant. We further demonstrate that yeast Dph3 (also known as KTI11), a CSL-type zinc finger protein, can bind iron and in the reduced state can serve as an electron donor to reduce the Fe-S cluster in Dph1-Dph2. Our study thus firmly establishes the functions for three of the proteins involved in eukaryotic diphthamide biosynthesis. For most radical SAM enzymes in bacteria, flavodoxins and flavodoxin reductases are believed to serve as electron donors for the Fe-S clusters. The finding that Dph3 is an electron donor for the Fe-S clusters in Dph1-Dph2 is thus interesting and opens up new avenues of research on electron transfer to Fe-S proteins in eukaryotic cells.

Scheme 1. Diphthamide Biosynthesis Pathway

Figure 1

Spectroscopic characterization of the [4Fe-4S] cluster in Dph1-Dph2 heterodimer. (A) UV–vis absorption spectra of anaerobically isolated (purple) and dithionite-reduced (red) Dph1-Dph2. (B) X-band EPR spectra of dithionite reduced Dph1-Dph2 at 12 K.

Figure 2

Reconstitution of Dph1-Dph2 activity with dithionite as the reductant. (A)HPLC analysis showing that reduced Dph1-Dph2 was sufficient in the SAM cleavage reaction, generating MTA with or without EF2. SAM was not cleaved without Dph1-Dph2 or dithionite. (B) Activity assay using carboxy ¹⁴C-SAM. Left panel, Coomassie blue-stained gel; right panel, the autoradiography. Lane 1: protein standard; Lane 2: reaction containing Dph1-Dph2, SAM, and dithionite; Lane 3: negative control containing Dph1-Dph2 and SAM but without dithionite.

Figure 3

Dph3 binds iron and is redox active. (A) UV–vis absorption spectrum of Dph3; Dph3 reduced by dithionite and reoxidized by air. (B) Dph3 can be reduced by NorW and NADH (monitored at 488 nm).

Figure 4

In vitro reconstitution of Dph1–Dph3 activity using carboxy-¹⁴C-SAM. The top panel shows the Coomassie-blue-stained gel; the bottom panel shows the autoradiography. All the reactions in lanes 2–7 contained EF2 and Dph1-Dph2. The presence of other reagents is indicated below each lane.