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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.

SUBMITTER: Park JH

PROVIDER: S-EPMC1324997 | biostudies-literature | 2006 Jan

REPOSITORIES: biostudies-literature

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

Park Jong-Hwan JH Aravind L L Wolff Edith C EC Kaevel Jörn J Kim Yeon Sook YS Park Myung Hee MH

Proceedings of the National Academy of Sciences of the United States of America 20051221 1

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 clo ...[more]

PMID: 16371467

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Project description:BACKGROUND: Plasmodium vivax is the most widespread human malaria parasite. However, genetic information about its pathogenesis is limited at present, due to the lack of a reproducible in vitro cultivation method. Sequencing of the Plasmodium vivax genome suggested the presence of a homolog of deoxyhypusine synthase (DHS) from P. falciparum, the key regulatory enzyme in the first committed step of hypusine biosynthesis. DHS is involved in cell proliferation, and thus a valuable drug target for the human malaria parasite P. falciparum. A comparison of the enzymatic properties of the DHS enzymes between the benign and severe Plasmodium species should contribute to our understanding of the differences in pathogenicity and phylogeny of both malaria parasites. RESULTS: We describe the cloning of a 1368 bp putative deoxyhypusine synthase gene (dhs) sequence from genomic DNA of P. vivax PEST strain Salvador I (Accession number AJ549098) after touchdown PCR. The corresponding protein was expressed and functionally characterized as deoxyhypusine synthase by determination of its specific activity and cross-reactivity to human DHS on a Western blot. The putative DHS protein from P. vivax displays a FASTA score of 75 relative to DHS from rodent malaria parasite, P. yoelii, and 74 relative to that from the human parasite, P. falciparum strain 3D7. The ORF encoding 456 amino acids was expressed under control of IPTG-inducible T7 promoter, and expressed as a protein of approximately 50 kDa (theoretically 52.7 kDa) in E. coli BL21 DE3 cells. The N-terminal histidine-tagged protein was purified by Nickel-chelate affinity chromatography under denaturing conditions. DHS with a theoretical pI of 6.0 was present in both eluate fractions. The specific enzymatic activity of DHS was determined as 1268 U/mg protein. The inhibitor, N-guanyl-1, 7-diaminoheptane (GC7), suppressed specific activity by 36-fold. Western blot analysis performed with a polyclonal anti-human DHS antibody revealed cross-reactivity to DHS from P. vivax, despite an amino acid identity of 44% between the proteins. CONCLUSION: We identify a novel DHS protein in the more benign malaria parasite,P. vivax, on the basis of specific enzymatic activity, cross-reactivity with a polyclonal antibody against human DHS, and amino acid identity with DHS homologs from the rodent malaria parasite, P. yoelii, and human P. falciparum strains.

Dataset Information

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

Publications

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

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