phosphorus-radioisotopes and diphthamide

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.

Topics: Amino Acid Sequence; Autoradiography; Biosynthetic Pathways; Carbon-Nitrogen Ligases; Chromatography, Liquid; Cloning, Molecular; DNA Primers; Electrophoresis, Polyacrylamide Gel; Genomics; Histidine; Humans; Ligases; Molecular Sequence Data; Molecular Structure; Phosphorus Radioisotopes; Plasmids; Rosaniline Dyes; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sequence Analysis, DNA; Species Specificity; Tandem Mass Spectrometry

Topics: Adenosine Diphosphate Ribose; Diphtheria Toxin; Histidine; NAD; Nucleoside Diphosphate Sugars; Peptide Elongation Factor 2; Peptide Elongation Factors; Phosphorus Radioisotopes; Protein Processing, Post-Translational; Radioisotope Dilution Technique