Diphthamide biosynthesis requires an organic radical generated by an iron-sulphur enzyme

Abstract

Archaeal and eukaryotic translation elongation factor 2 contain a unique post-translationally modified histidine residue called diphthamide, which is the target of diphtheria toxin. The biosynthesis of diphthamide was proposed to involve three steps, with the first being the formation of a C-C bond between the histidine residue and the 3-amino-3-carboxypropyl group of S-adenosyl-l-methionine (SAM). However, further details of the biosynthesis remain unknown. Here we present structural and biochemical evidence showing that the first step of diphthamide biosynthesis in the archaeon Pyrococcus horikoshii uses a novel iron-sulphur-cluster enzyme, Dph2. Dph2 is a homodimer and each of its monomers can bind a [4Fe-4S] cluster. Biochemical data suggest that unlike the enzymes in the radical SAM superfamily, Dph2 does not form the canonical 5'-deoxyadenosyl radical. Instead, it breaks the C(gamma,Met)-S bond of SAM and generates a 3-amino-3-carboxypropyl radical. Our results suggest that P. horikoshii Dph2 represents a previously unknown, SAM-dependent, [4Fe-4S]-containing enzyme that catalyses unprecedented chemistry.

Figure 1

The structure of diphthamide and its proposed biosynthesis pathway. The diphthamide residue is the target of bacterial ADP-ribosyltransferases, diphtheria toxin and Pseudomonas exotoxin A.

Figure 2

Structure of PhDPH2 homodimer. The PhDph2 homodimer is shown in the ribbon diagram with one monomer in dark color and the other in light color. Each monomer is also color by secondary structure. The three conserved cysteine residues for each monomer are shown in the stick representation.

Figure 3

In vitro reconstitution of PhDph2 activity. a, Activity assay using carboxy-¹⁴C SAM. Top panel shows the Coomassie blue-stained gel; bottom panel shows the autoradiography. Lane 1: Protein standard; 2: Blank lane; 3: PhEF2 + SAM, negative control; 4: PhDph2 + SAM, negative control; 5: PhEF2 H600A + PhDph2 + SAM, negative control; 6: PhEF2 + PhDph2 + SAM + dithionite; 7: PhEF2 + PhDph2 + SAM, no dithionite, negative control. b, The MALDI-MS spectra of PhEF2 unmodified (top) and modified by PhDph2 in an in vitro reaction (bottom).

Figure 4

Spectroscopic characterization of the [4Fe-4S] cluster in PhDph2. a, UV-vis absorption spectra of anaerobically-isolated and dithionite-reduced PhDph2. b, X-band EPR spectra of dithionite-reduced PhDph2 at different temperature. c, 4.2-K/53-mT Mössbauer spectrum of anaerobically-isolated ⁵⁷Fe-labeled PhDph2 expressed in E. coli. d, Structure of PhDph2 with [4Fe-4S] cluster.

Figure 5

Identification of SAM-derived small molecule products in PhDph2-catalyzed reactions. a, HPLC analysis of reaction products suggests PhDph2 does not form 5′-deoxyadenosine. b, ¹H-NMR showing that ABA and HSA formed in the reaction when PhEF2 was not present, but not in control reaction where PhDph2 was not present. The peaks from HSA and ABA are marked by blue and magenta arrows, respectively. c, Detection of dansylated reaction products by LCMS. The MS traces (total ion counts and ion counts for specific compounds) were shown for the reaction with PhDph2, control reaction without PhDph2, and ABA and HSA standards.

Figure 6

The proposed reaction mechanism for PhDph2. The formation of ABA and HSA can be best explained by a 3-amino-3-carboxypropyl radical intermediate. The radical can be generated by electron transfer from the [4Fe-4S] cluster, similar to the generation of 5′-deoxyadenosyl radical in other radical SAM enzymes. In the presence of PhEF2, the radical will react with PhEF2 to form the modified PhEF2 product. In the absence of PhEF2, the radical can either abstract a hydrogen atom to form ABA or be quenched by dithionite to give HSA.