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(epsilon)-(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 alpha-helical pair (HEAT motif) organized in a symmetrical dyad. The predicted structure is unrelated to the double-stranded beta-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.

Fig. 1.

Multiple alignment of DOHH from diverse eukaryotic taxa. The individual HEAT repeats are numbered and shown above the alignment. The two sets of four HEAT repeats, which represent the last internal duplication in this protein, are shown. The secondary structure prediction (H represents α-helix and E an extended configuration) derived by using the jpred program (32) is shown above the alignment. The four characteristic HE motifs are highlighted in reverse shading. The coloring reflects the conservation profile at the 95% consensus. Absolutely conserved residues (capital letter, gray), hydrophobic residues (h: ACFILMVWY), big residues (b: FILMYWKERQ), alcoholic residues (o: T,S), small residues (s: AGSVCDN), tiny subset residues (t: GAS), polar residues (p: STEDKRNQHC), and charged residues (c: DEKHR) are shown in different colors.

Fig. 2.

Comparison of growth, hypusine synthesis, and hypusine/deoxyhypusine content in DOHH-null and parent strains. The S. cerevisiae DOHH-null strain and the parental strain, BY4743, were cultured at 28°C. (A) SDS/PAGE after labeling with [1,8-³H]spermidine·HCl. (B) Ion exchange chromatographic separation of labeled hypusine and deoxyhypusine from labeled eIF5A proteins. (C) Growth curves. (D) Fluorometric detection of hypusine and deoxyhypusine in total cell protein (29).

Fig. 3.

Expression, purification, and activity determination of human and yeast GST-DOHH and free DOHH enzymes. DOHH proteins were expressed in BL21(DE3) cells as described in Materials and Methods. (A) SDS/PAGE of cell lysate (≈50 μg of protein). (B) DOHH activity assays of the lysate (2 μg of total protein). (C) SDS/PAGE of purified proteins. Lanes: 1–3, yeast enzyme; 4–6, human enzyme; 1 and 4, purified GST fusion proteins before thrombin treatment; 2 and 5, after thrombin treatment of GST-DOHH; 3 and 6, DOHH released after thrombin treatment of GSH-Sepharose-bound GST-DOHH. (D) DOHH activity assays with 0.02, 0.05, 0.1, and 0.2 μg of purified enzymes.

Fig. 4.

Overproduction of hypusine-containing eIF5A in 293T cells by cotransfection with three vectors encoding eIF5A, DHS, and DOHH. The 293T cells were transfected with empty pCEFL vector (lane 1), p3XFLAG-CMV-7.1/heIF5A-1 alone (lane 2), together with pCEFL/hDHS (lane 3), or with pCEFL/hDHS plus pCEFL/hDOHH (lane 4). (A) Western blot with eIF5A-1 Ab. This Ab recognizes all three forms of eIF5A-1: the precursor [eIF5A(Lys)], the intermediate [eIF5A-1(Dhp)], and the mature eIF5A-1 (hypusine form) equally well. (B) Fluorogram of SDS gel of proteins of cells cultured with [³H]spermidine. (C) Content of [³H]hypusine and [³H]deoxyhypusine in the labeled proteins.

Fig. 5.

Comparison of the predicted structure of DOHH with those of two DSBH dioxygenases. (Upper) The predicted structure of DOHH (Left), numbered according to the human protein, and a structural model (Right) based on the known x-ray structure of YibA (Protein Data Bank ID code 1OYZ). These models of DOHH structure are only approximations for its actual structure, and alternative modes of metal chelation and configuration of the HEAT repeats are possible (see text). (Lower) Topological diagrams (27) of two DSBH dioxygenases: AlkB and prolyl-4-hydroxylase (Protein Data Bank ID code 1E5S).