Mechanistic understanding of Pyrococcus horikoshii Dph2, a [4Fe-4S] enzyme required for diphthamide biosynthesis

Abstract

Diphthamide, the target of diphtheria toxin, is a unique posttranslational modification on eukaryotic and archaeal translation elongation factor 2 (EF2). The proposed biosynthesis of diphthamide involves three steps and we have recently found that in Pyrococcus horikoshii (P. horikoshii), the first step uses an S-adenosyl-L-methionine (SAM)-dependent [4Fe-4S] enzyme, PhDph2, to catalyze the formation of a C-C bond. Crystal structure shows that PhDph2 is a homodimer and each monomer contains three conserved cysteine residues that can bind a [4Fe-4S] cluster. In the reduced state, the [4Fe-4S] cluster can provide one electron to reductively cleave the bound SAM molecule. However, different from classical radical SAM family of enzymes, biochemical evidence suggest that a 3-amino-3-carboxypropyl radical is generated in PhDph2. Here we present evidence supporting that the 3-amino-3-carboxypropyl radical does not undergo hydrogen abstraction reaction, which is observed for the deoxyadenosyl radical in classical radical SAM enzymes. Instead, the 3-amino-3-carboxypropyl radical is added to the imidazole ring in the pathway towards the formation of the product. Furthermore, our data suggest that the chemistry requires only one [4Fe-4S] cluster to be present in the PhDph2 dimer.

Figure 1

Diphthamide biosynthesis pathway.

Figure 2

Two possible mechanisms for PhDph2-catalyzed reaction. a. One [4Fe-4S] cluster per PhDph2 dimer is sufficient for the reaction and the ACP radical adds to the imidazole ring; b. Two [4Fe-4S] clusters per PhDph2 dimer are required for the reaction. The ACP radical abstracts a hydrogen atom from the imidazole ring. As a consequence, one reaction needs two SAM with one ACP transferred to PhEF and the other one released as 2-aminobutyric acid.

Figure 3

Spectroscopy characterization of PhDph2 mutants. a. UV-Vis spectroscopy of as purified PhDph2 wild type (black line), single mutants: C59A (red line), C163A (blue line), C287A (green line) and double mutant(pink line); b. UV-Vis spectroscopy of single mutant PhDph2 C59A with and without dithionite; c. EPR spectra of wild type PhDph2 and its single mutants in the reduced [4Fe – 4S]¹⁺ state at 12 K. The principal values of g-factor for the main spectral component (shown for wild type and C163A with arrows of corresponding color) are: WT: 2.03, 1.92, 1.86; C287A: 2.01, 1.94, 1.88. S59A: 2.03, 1.94, 1.90; C163A: 2.11, 1.91, 1.793;

Figure 4

Activity assay of PhDph2 single mutants monitored by autoradiography and HPLC. a. ¹⁴C- labeling of PhEF2 by PhDp2 wild type and three single mutants. The reactions contain PhEF2, PhDph2 (WT or mutants), SAM, and dithionite. Left panel shows the Coomassie Blue-stained gel and right panel shows the autoradiography. b. Reaction product methylthioadenosine (MTA) formation was detected by HPLC. SAM was eluted at 2 min and MTA was eluted at 21.5 min.

Figure 5

Tandem purification to get different PhDph2 dimers. a. Diagram showing the tandem purification strategy to get different PhDph2 dimers; b. Tandem purification of PhDph2 dimers: left, elutions from Ni-NTA purification by using different concentrations of imidazole solutions to get PhDph2 (WT-His₆) and PhDph2 (DM-GST: WT-His₆) mixture; Right, purification of PhDph2 (DM-GST), PhDph2 (DM-GST: WT-His₆), and PhDph2 (WT-His₆). PhDph2 (WT-His₆). c. Purified phDph2 dimers. The PhDph2 (WT-His₆) shown here was further purified by heating at 95 °C.

Figure 6

Stability of PhDph2 homodimer and PhDph2 (DM-GST: WT-His₆) heterodimer. a. PhDph2 (DM-GST) cannot bind to Ni resin, it was found in the flow through of Ni-affinty purification but not in the elutions. b. Stability test. Left of the protein marker: PhDph2 (WT-His₆) and (DM-GST) were mixed, incubated for 90 min, and then purified with Ni-resin. PhDph2 (DM-GST) was found in flow through; PhDph2 (DM-GST: WT-His₆) and PhDph2 (WT-His₆) were eluted from Ni-resin by 150 mM and 200 mM imidazole; Right of the protein marker: PhDph2 (DM-GST: WT-His₆) heterodimer from tandem purification was incubated with Ni-resin then eluted with 150 mM imidazole. No homodimer was found in the flow through.

Figure 7

Activity assay of PhDph2 heterodimer. a. ¹⁴C-labeling of PhEF2 catalyzed by PhDph2 (WT-His₆), PhDph2 (DM-GST: WT-His₆), PhDph2 (DM: WT-His₆), and PhDph2 (DM-GST). The PhDph2 (DM: WT-His₆) was obtained by TEV digest of PhDph2 (DM-GST: WT-His₆) to remove the GST tag. The left panel shows the Coomassie blue-stained gel and the right panel shows the autoradiography. b. The formation of MTA was detected by HPLC.

Figure 8

Detection of dansylated ABA (2-aminobutyric acid) by LCMS. Left panel shows the MS traces and right panel shows the LC traces. Components used in different reactions were labeled in the figure. ABA was only formed in the reaction with PhDph2 but without PhEF2.