Abstract
Deoxyhypusine synthase catalyzes the first step in the posttranslational synthesis of an unusual amino acid, hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine), in the eukaryotic translation initiation factor 5A (eIF-5A) precursor protein. The null mutation in the single copy gene, yDHS, encoding deoxyhypusine synthase results in the loss of viability in the yeast Saccharomyces cerevisiae. Upon depletion of deoxyhypusine synthase, and consequently of eIF-5A, cessation of growth was accompanied by a marked enlargement of cells, suggesting a defect in cell cycle progression or in cell division. Two residues of the yeast enzyme, Lys308 and Lys350, corresponding to Lys287 and Lys329, respectively, known to be critical for the activity of the human enzyme, were targeted for site-directed mutagenesis. The chromosomal ydhs null mutation was complemented by the plasmid-borne yDHS wild-type gene, but not by mutated genes encoding inactive proteins, including that with Lys350-->Arg substitution or with substitutions at both Lys308 and Lys350. The mutated gene ydhs (K308R) encoding a protein with diminished activities (< 1% of wild type) could support growth but only to a very limited extent. These findings provide strong evidence that the hypusine modification is indeed essential for the survival of S. cerevisiae and imply a vital function for eIF-5A in all eukaryotes.
Highlights
The biosynthesis of the unusual amino acid, hypusine (N⑀(4-amino-2-hydroxybutyl)lysine), represents a novel posttranslational modification that occurs exclusively in one cellular protein, the precursor of eukaryotic translation initiation factor 5A1 [1, 2]
We and others identified YHR068w in S. cerevisiae chromosome VIII as a candidate gene for yeast deoxyhypusine synthase by searching for sequences in the GenBank data base that matched the partial amino acid sequences determined for the enzymes from rat [4], human [7], or N. crassa cDNA sequences [11]
The identity of the deoxyhypusine synthase gene was verified by demonstrating enzymatic activity after expression of its open reading frame (ORF) in E. coli [12]
Summary
The biosynthesis of the unusual amino acid, hypusine (N⑀(4-amino-2-hydroxybutyl)lysine), represents a novel posttranslational modification that occurs exclusively in one cellular protein, the precursor of eukaryotic translation initiation factor 5A (eIF-5A)1 [1, 2]. In the first step of hypusine synthesis, deoxyhypusine synthase catalyzes transfer of the butylamine moiety of the polyamine spermidine to the ⑀-amino group of a specific lysine residue of eIF-5A precursor (Lys in the yeast proteins) to form a deoxyhypusine (N⑀-(4-aminobutyl)lysine) residue [3, 4]. In recent studies with human deoxyhypusine synthase, we have identified an active site lysine residue, Lys329, that is involved in enzyme-substrate intermediate formation [13]. Hypusine is ubiquitous in eukaryotes, occurring at one highly conserved residue of eIF-5A The assignment of this protein as a putative translation initiation factor was based on its in vitro ability to stimulate methionyl puromycin synthesis [14]. Our results presented here show that the activity of deoxyhypusine synthase, as well as the gene product, is required for yeast viability
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