Chemogenomic approach identified yeast YLR143W as diphthamide synthetase

Abstract

Many genes are of unknown functions in any sequenced genome. A combination of chemical and genetic perturbations has been used to investigate gene functions. Here we present a case that such "chemogenomics" information can be effectively used to identify missing genes in a defined biological pathway. In particular, we identified the previously unknown enzyme diphthamide synthetase for the last step of diphthamide biosynthesis. We found that yeast protein YLR143W is the diphthamide synthetase catalyzing the last amidation step using ammonium and ATP. Diphthamide synthetase is evolutionarily conserved in eukaryotes. The previously uncharacterized human gene ATPBD4 is the ortholog of yeast YLR143W and fully rescues the deletion of YLR143W in yeast.

Fig. 1.

Biosynthetic pathway of diphthamide in eukaryotes.

Fig. 2.

The eight genes having highest cofitness values to (A) YBR246W deletion strain and (B) DPH1, 2, 4, and 5 deletion strains as a whole group. The rank of each correlation is labeled to the bottom right of each circle.

Fig. 3.

Diphthine accumulates in Δylr143w yeast strain. (A) Labeling of eEF-2 purified from various strains using Rh-NAD and DT. The strains used are specified above each lane. In lanes 1–4, low DT concentration (0.1 μM) was used. In lanes 5–8, high DT concentration (10 μM) was used. (B) Diphtheria toxin sensitivity assay. The strains used are specified at the top. Plasmids used in the transformation are listed to the right. The cells were grown on 2% galactose. Each row represents a serial dilution from left to the right. (C) The mass spectrum of the eEF-2 peptide containing diphthine from the Δylr143w strain. (D) Ectopic expression of YLR143W or human ATPBD4 restores diphthamide biosynthesis in the Δylr143w strain. The plasmids used are shown above each lane. HsATPBD4 is the human ATPBD4. PhATPBD4 is the ortholog from P. horikoshii (PH1257). Low DT concentration (0.1 μM) was used in all lanes.

Fig. 4.

YLR143W uses ATP and NH₄⁺ for the amidation reaction. (A) YLR143W in vitro reconstitution detected using DT (0.1 μM) and Rh-NAD. All reactions contain eEF-2 purified from the Δylr143w strain. Purified YLR143W and small molecule substrates used are indicated above each lane. (B) AMP is formed in the diphthine amidation reaction. All lanes contained [α-³²P]-ATP. ADP and AMP standards in lanes 2 and 3 were generated enzymatically from ATP. (C) Proposed reaction pathway for YLR143W-catalyzed diphthine amidation reaction.