Molecular Cloning, Expression, and Structural Prediction of Deoxyhypusine Hydroxylase: A HEAT-Repeat-Containing Metalloenzyme

• Published in: Proceedings of the National Academy of Sciences - PNAS 2006-01, Vol.103 (1), p.51-56
• Main Authors: Park, Jong-Hwan, Aravind, L., Wolff, Edith C., Kaevel, Jörn, Kim, Yeon Sook, Park, Myung Hee
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs - genetics; Amino Acid Sequence; Amino acids; Archaea; Biochemistry; Biological Sciences; Cell Line; Chromatography, Ion Exchange; Cloning, Molecular; Computational Biology; DNA Primers; Enzymes; Eukaryotes; Eukaryotic cells; Eukaryotic Translation Initiation Factor 5A; Gene Expression; Gene Library; Genetic vectors; Goods and services tax; Humans; Iron - metabolism; Libraries; Mixed Function Oxygenases - chemistry; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - metabolism; Models, Molecular; Molecular Sequence Data; Open reading frames; Peptide Initiation Factors - genetics; Protein precursors; Proteins; RNA-Binding Proteins - genetics; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Sequence Alignment; Yeast; Yeasts
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0509348102

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(ll)- 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.