Kee Ryeon Kang

Deoxyhypusine synthase activity is essential for cell viability in the yeast Saccharomyces cerevisiae.

Deoxyhypusine synthase catalyzes the first step in the posttranslational synthesis of an unusual amino acid, hypusine (N ε-(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 yDHSwild-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.

Posttranslational synthesis of hypusine: evolutionary progression and specificity of the hypusine modification

Summary.A naturally occurring unusual amino acid, hypusine [Nɛ-(4-amino-2-hydroxybutyl)-lysine] is a component of a single cellular protein, eukaryotic translation initiation factor 5A (eIF5A). It is a modified lysine with structural contribution from the polyamine spermidine. Hypusine is formed in a novel posttranslational modification that involves two enzymes, deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). eIF5A and deoxyhypusine/hypusine modification are essential for growth of eukaryotic cells. The hypusine synthetic pathway has evolved in eukaryotes and eIF5A, DHS and DOHH are highly conserved, suggesting maintenance of a fundamental cellular function of eIF5A through evolution. The unique feature of the hypusine modification is the strict specificity of the enzymes toward its substrate protein, eIF5A. Moreover, DHS exhibits a narrow specificity toward spermidine. In view of the extraordinary specificity and the requirement for hypusine-containing eIF5A for mammalian cell proliferation, eIF5A and the hypusine biosynthetic enzymes present new potential targets for intervention in aberrant cell proliferation.

DEOXYHYPUSINE HYDROXYLASE IS A Fe(II)-DEPENDENT, HEAT-REPEAT ENZYME: IDENTIFICATION OF AMINO ACID RESIDUES CRITICAL FOR Fe(II) BINDING AND CATALYSIS

Deoxyhypusine hydroxylase (DOHH) catalyzes the final step in the post-translational synthesis of hypusine (Nϵ-(4-amino-2-hydroxybutyl)lysine) in eIF5A. DOHH is a HEAT-repeat protein with eight tandem helical hairpins in a symmetrical dyad. It contains two potential iron coordination sites (one on each dyad) composed of two strictly conserved His-Glu motifs. The purified human recombinant DOHH was a mixture of active holoenzyme containing 2 mol of iron/mol of DOHH and inactive metal-free apoenzyme. The two species could be distinguished by their different mobilities upon native gel electrophoresis. The DOHH apoenzyme exhibited markedly reduced levels of iron and activity. DOHH activity could be restored only by the addition of Fe2+ to the apoenzyme but not by other metals including Cd2+,Co2+,Cr2+,Cu2+,Mg2+,Mn2+,Ni2+, and Zn2+. The role of the strictly conserved His-Glu residues was evaluated by site-directed mutagenesis. Substitution of any single amino acid in the four His-Glu motifs with alanine abolished the enzyme activity. Of these eight alanine substitutions, six, including H56A, H89A, E90A, H207A, H240A, and E241A, caused a severe reduction in the iron content. Our results provide strong evidence that Fe(II) is the active-site-bound metal critical for DOHH catalysis and that the strictly conserved His-Glu motifs are essential for iron binding and catalysis. Furthermore, the iron to DOHH stoichiometry and dependence of iron binding on each of the four conserved His-Glu motifs suggest a binuclear iron mediated reaction mechanism, distinct from that of other Fe(II)-dependent protein hydroxylases, such as prolyl 4-hydroxylase or lysyl hydroxylases.

The polyamine-derived amino acid hypusine: its post-translational formation in eIF-5A and its role in cell proliferation

SummaryThe unusual amino acid hypusine [Nε-(4-amino-2-hydroxybutyl)lysine] is a unique component of one cellular protein, eukaryotic translation initiation factor 5A (eIF-5A, old terminology, eIF-4D). It is formed posttranslationally and exclusively in this protein in two consecutive enzymatic reactions, (i) modification of a single lysine residue of the eIF-5A precursor protein by the transfer of the 4-aminobutyl moiety of the polyamine spermidine to itsε-amino group to form the intermediate, deoxyhypusine [Nε-(4-aminobutyl)lysine] and (ii) subsequent hydroxylation of this intermediate to form hypusine. The amino acid sequences surrounding the hypusine residue are strictly conserved in all eukaryotic species examined, suggesting the fundamental importance of this amino acid throughout evolution. Hypusine is required for the activity of eIF-5Ain vitro. There is strong evidence that hypusine and eIF-5A are vital for eukaryotic cell proliferation. Inactivation of both of the eIF-5A genes is lethal in yeast and the hypusine modification appears to be a requirement for yeast survival (Schnier et al., 1991 [Mol Cell Biol 11: 3105–3114]; Wöhl et al., 1993 [Mol Gen Genet 241: 305–311]). Furthermore, inhibitors of either of the hypusine biosynthetic enzymes, deoxyhypusine synthase or deoxyhypusine hydroxylase, exert strong anti-proliferative effects in mammalian cells, including many human cancer cell lines. These inhibitors hold potential as a new class of anticancer agents, targeting one specific eukaryotic cellular reaction, hypusine biosynthesis.

Specificity of the deoxyhypusine hydroxylase-eukaryotic translation initiation factor (eIF5A) interaction: identification of amino acid residues of the enzyme required for binding of its substrate, deoxyhypusine-containing eIF5A.

Deoxyhypusine hydroxylase (DOHH) is a novel metalloenzyme that catalyzes the final step of the post-translational synthesis of hypusine (Nϵ-(4-amino-2-hydroxybutyl)lysine) in the eukaryotic translation initiation factor 5A (eIF5A). Hypusine synthesis is unique in that it occurs in only one protein, denoting the strict specificity of the modification enzymes toward the substrate protein. The specificity of the interaction between eIF5A and DOHH was investigated using human eIF5A (eIF5A-1 isoform) and human recombinant DOHH. DOHH displayed a strong preference for binding the deoxyhypusine-containing form of eIF5A, over the eIF5A precursor or the hypusine-containing eIF5A, indicating a role for the deoxyhypusine residue in binding. In addition to the deoxyhypusine residue, a large portion of the eIF5A polypeptide (>20-90 amino acids) is required for effective modification by DOHH. We have identified the amino acid residues of DOHH that are critical for substrate binding by alanine substitution of 36 conserved amino acid residues. Of these, alanine substitution at Glu57, Glu90, Glu208, Glu241, Gly63, or Gly214 caused a severe impairment in eIF5A(Dhp) binding, with a complete loss of binding and activity in the E57A and E208A mutant enzymes. Only aspartate substitution mutants, E57D or E208D, retained partial activity and substrate binding, whereas alanine, glutamine, or asparagine mutants did not. These findings support a proposed model of DOHH-eIF5A binding in which the amino group(s) of the deoxyhypusine side chain of the substrate is primarily anchored by γ-carboxyl groups of Glu57 and Glu208 at the DOHH active site.

Protein kinase CK2 phosphorylates and interacts with deoxyhypusine synthase in HeLa cells

Deoxyhypusine is a modified lysine and formed posttranslationally to be the eukaryotic initiation factor eIF5A by deoxyhypusine synthase, employing spermidine as butylamine donor. Subsequent hydroxylation of this deoxyhypusine-containing intermediate completes the maturation of eIF5A. The previous report showed that deoxyhypusine synthase was phosphorylated by PKC in vivo and the association of deoxyhypusine synthase with PKC in CHO cells was PMA-, and Ca(2+)/phospholipid-dependent. We have extended study on the phosphorylation of deoxyhypusine synthase by protein kinase CK2 in order to define its role on the regulation of eIF5A in the cell. The results showed that deoxyhypusine synthase was phosphorylated by CK2 in vivo as well as in vitro. Endogenous CK2 in HeLa cells and the cell lysate was able to phosphorylate deoxyhypusine synthase and this modification is enhanced or decreased by the addition of CK2 effectors such as polylysine, heparin, and poly(Glu, Tyr) 4:1. Phosphoamino acid analysis of this enzyme revealed that deoxyhypusine synthase is mainly phosphorylated on threonine residue and less intensely on serine. These results suggest that phosphorylation of deoxyhypusine synthase is CK2-dependent cellular event as well as PKC-mediated effect. However, there were no observable changes in enzyme activity between the phosphorylated and unphosphorylated forms of deoxyhypusine synthase. Taken together, besides its established function in hypusine modification involving eIF5A substrate, deoxyhypusine synthase and its phosphorylation modification may have other independent cellular functions because of versatile roles of deoxyhypusine synthase.