Molecular cloning, expression, and structural prediction of deoxyhypusine hydroxylase: A HEAT-repeat-containing metalloenzyme

Abstract

The eukaryotic initiation factor 5A (eIF5A), a factor essential for eukaryotic cell proliferation, is the only cellular protein containing the polyamine-derived amino acid hypusine [N -(4-amino-2-hydroxybutyl)lysine]. Hypusine is formed in a posttranslational modification that involves two sequential enzymatic steps catalyzed by deoxyhypusine synthase and deoxyhypusine hydroxylase (DOHH). By screening a Saccharomyces cerevisiae GST-ORF library for expression of DOHH activity, we have cloned YJR070C as the gene encoding DOHH and identified the human homolog DOHH gene, HLRC1. Purified recombinant yeast and human DOHH enzymes effectively catalyzed hydroxylation of the deoxyhypusine residue in the eIF5A intermediate. Overexpression of human DOHH along with eIF5A precursor and deoxyhypusine synthase was required for overproduction of mature, hypusine-containing eIF5A in 293T and other mammalian cells. The Saccharomyces cerevisiae strain with deletion of YJR070C contained only deoxyhypusine but no hypusine, indicating that YJR070C was the single DOHH gene in this organism. One highly conserved DOHH homolog gene is found in a variety of eukaryotes from yeast to human. Sequence and structural analyses reveal that DOHH belongs to a family of HEAT-repeat-containing proteins, consisting of eight tandem repeats of an ␣-helical pair (HEAT motif) organized in a symmetrical dyad. The predicted structure is unrelated to the double-stranded ␤-helix type structures of the Fe(II)-and 2-oxoacid-dependent dioxygenases, such as collagen prolyl or lysyl hydroxylases. However, metal coordination sites composed of four strictly conserved histidine-glutamate sequences were identified, suggesting that DOHH enzymes have convergently evolved an iron-dependent hydroxylation mechanism. eukaryotic initiation factor 5A ͉ hypusine ͉ YJR070C

Human deoxyhypusine hydroxylase, an enzyme involved in regulating cell growth, activates O 2 with a nonheme diiron center

“…hDOHH appears not to follow this pattern. Instead, hDOHH has a pair of conserved HEX 31 HE sequences that are symmetrically located near the C and N termini, which possibly provide the metal binding ligands (9). On the basis of homology modeling and circular dichroism experiments, the overall structure of hDOHH has been proposed to contain a dyad of 4 consecutive ␣-hairpins (called a HEAT repeat motif) surrounding the active site (9).…”

“…An estimated iron-to-holoprotein stoichiometry of 2 is observed (8). Sequence examination, homology modeling, and mutagenesis experiments suggest 2 possible iron binding sites consisting of histidine and carboxylate ligands (8,9). Thus at first glance, hDOHH appears to resemble members of the superfamily of bacterial diiron multicomponent monooxygenases, like methane or toluene monooxygenase, that use nonheme diiron centers to activate dioxygen for the hydroxylation of hydrocarbons (10-12).…”

Evaluation of Difluoromethylornithine for the Chemoprevention of Barrett's Esophagus and Mucosal Dysplasia

“…Of note, DFMO was shown to downregulate cyclin E expression in human breast cancer cells (49). Other genes that influence cell proliferation and were modulated by DFMO include HLRC1 (MGC4293), also known as the DOHH gene (33,34), and LOC134492 whose NudCL2 product localized to the centrosome, spindle poles, and kinetochores during mitosis (35). DFMO has been shown to suppress mucosal polyamines coincident with a reduction in epithelial cell proliferation in human rectal mucosa (19).…”

“…Hypusine is a polyamine-derived amino acid that is formed in the eukaryotic translation initiation factor 5A (eIF5A) by a posttranslational modification that involves 2 enzymatic steps (33). In the second step, DOHH converts the deoxyhypusine-containing intermediate to the hypusine-containing mature eIF5A (34). The eIF5A protein and deoxyhypusine/hypusine modification are essential for eukaryotic cell proliferation.…”

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

“…In 1995, we and others reported purification and cloning of the first step enzyme, deoxyhypusine synthase (Joe et al, 1995;Tao and Chen, 1995). The identity of the second step enzyme, deoxyhypusine hydroxylase, remained elusive, until our recent identification of the previously reported LIA1 (ligand of eIF5A) (Thompson et al, 2003) as the DOHH gene, its cloning and characterization Park et al, 2006). eIF5A and its deoxyhypusine/hypusine modification are vital for eukaryotic cell proliferation (Gerner et al, 1986;Byers et al, 1992;Chen and Liu, 1997;Chattopadhyay et al, 2003;Park, 2006).…”