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

Abstract

Deoxyhypusine hydroxylase is the key enzyme in the biosynthesis of hypusine containing eukaryotic translation initiation factor 5A (eIF5A), which plays an essential role in the regulation of cell proliferation. Recombinant human deoxyhypusine hydroxylase (hDOHH) has been reported to have oxygen-and iron-dependent activity, an estimated iron/holoprotein stoichiometry of 2, and a visible band at 630 nm responsible for the blue color of the as-isolated protein. EPR, Mö ssbauer, and XAS spectroscopic results presented herein provide direct spectroscopic evidence that hDOHH has an antiferromagnetically coupled diiron center with histidines and carboxylates as likely ligands, as suggested by mutagenesis experiments. Resonance Raman experiments show that its blue chromophore arises from a ( -1,2-peroxo)diiron(III) center that forms in the reaction of the reduced enzyme with O 2, so the peroxo form of hDOHH is unusually stable. Nevertheless we demonstrate that it can carry out the hydroxylation of the deoxyhypusine residue present in the elF5A substrate. Despite a lack of sequence similarity, hDOHH has a nonheme diiron active site that resembles both in structure and function those found in methane and toluene monooxygenases, bacterial and mammalian ribonucleotide reductases, and stearoyl acyl carrier protein ⌬ 9 -desaturase from plants, suggesting that the oxygen-activating diiron motif is a solution arrived at by convergent evolution. Notably, hDOHH is the only example thus far of a human hydroxylase with such a diiron active site.H ypusine is an unusual, but highly conserved, amino acid that is found only in the eukaryotic translational initiation factor 5A (eIF5A), a protein that regulates cell proliferation (1, 2). The biosynthesis of eIF5A involves a posttranslational modification of the eIF5A precursor, where a lysine residue is first modified to deoxyhypusine (Dhp) by deoxyhypusine synthase (DHS) and then the nascent Dhp is hydroxylated by deoxyhypusine hydroxylase (DOHH) to form hypusine (Hpu) (Scheme 1) (1, 2). The importance of hypusine and these 2 enzymes has been shown by several studies where depletion of spermidine (3) or inhibition of either DHS or DOHH (4, 5) leads to a decrease of hypusinecontaining eIF5A [eIF5A(Hpu)] and inhibition of eukaryotic cell growth. Consequently, these results suggest that eIF5A and DOHH could be promising targets for antitumor (6) and anti-HIV-1 therapies (7).The hydroxylase activity of recombinant human DOHH (hDOHH) has been shown to depend on Fe(II) and not on any other physiologically relevant divalent metal ion. 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 hydroxylat...

A family of diiron monooxygenases catalyzing amino acid beta-hydroxylation in antibiotic biosynthesis

“…However, the C-terminal closely resembles the metallo β-lactamase family of enzymes. The overall αββα fold of metallo-β−lactamases represents a significant structural departure from the 4-α-helix bundle core structure that has been found for all oxygenases with diiron clusters in previous studies (18) with the possible exception of human deoxyhypusine hydroxylase, which may use a series of α-hairpin turns to support the cluster (35).…”

Cytosolic iron chaperones: Proteins delivering iron cofactors in the cytosol of mammalian cells

“…In cells lacking PCBP1 or PCBP2, deoxyhypusine accumulates and hypusine levels fall, indicating a loss of DOHH activity. Two iron ions in the active site of DOHH form a peroxo bridge to activate oxygen for this reaction (34). The apo-and holo-forms of the enzyme can be distinguished by their distinct electrophoretic mobilities.…”

Iron chaperones PCBP1 and PCBP2 mediate the metallation of the dinuclear iron enzyme deoxyhypusine hydroxylase

“…In the second step, DOHH hydroxylates the Dhp to form Hpu. DOHH is a HEAT-repeat enzyme that contains a peroxo-linked, dinuclear iron center in which the iron ions are coordinated by two sets of conserved histidine and glutamate residues (23,25,26). Hpu synthesis occurs only on a single lysine residue on a single protein, eIF5A.…”

“…Recombinant DOHH is purified from Escherichia coli in two forms, one that migrates slowly in native gels and contains no iron and no enzymatic activity (apo-DOHH) and one that migrates more rapidly and contains both iron and enzymatic activity (holo-DOHH) (23,31). Furthermore, only Fe(II), and not other divalent metals, can convert apo-DOHH to the more compact holo form, which contains a peroxo-iron bridge, in vitro (23,26). We confirmed that the faster-migrating, holo form of DOHH corresponds to the iron-and oxygen-bound enzyme by noting its appearance when both iron and air are present but its absence when iron is added in the absence of oxygen (Fig.…”