An unusual amino acid, hypusine, which occurs in only one cellular protein, eukaryotic translation isolation factor 5A (eIF-5A), is intimately involved in cell proliferation. Hypusine biosynthesis occurs by way of two sequential post-translational modification reactions: i) deoxyhypusine synthesis by deoxyhypusine synthase and ii) deoxyhypusine hydroxylation by deoxyhypusine hydroxylase. We have purified the first enzyme, deoxyhypusine synthase, to homogeneity after about 100,000-fold enrichment from rat testis. The purified enzyme displays a remarkably narrow specificity toward its substrates, spermidine, NAD, and the eIF-5A precursor protein and catalyzes deoxyhypusine synthesis in the complete reaction mixture. In the absence of the substrate protein, however, it carries out a partial reaction, the NAD-dependent cleavage of spermidine. The enzyme exists as a tetramer of 42 kDa subunits, with a pI of 4.75. Using partial amino acid sequences from the rat testis enzyme, we were able to identify YHRO68W of Saccharomyces cerevisiae chromosome VIII as a gene for deoxyhypusine synthase. We have also cloned human cDNAs encoding a full-length deoxyhypusine synthase by immunoscreening of a HeLa cell cDNA library. After overexpression of the human deoxyhypusine synthase cDNA or the yeast cDNA of YHRO68W in E. coli, we purified the recombinant enzymes and characterized their physical and enzymatic properties. Deletion, insertion and site-directed mutagenesis studies are underway to gain insights into the active site structure and the structure-function relationship of this important enzyme.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Intramural Research (Z01)
Project #
1Z01DE000608-02
Application #
5201850
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
1995
Total Cost
Indirect Cost
Name
National Institute of Dental & Craniofacial Research
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Chattopadhyay, Manas K; Park, Myung Hee; Tabor, Herbert (2008) Hypusine modification for growth is the major function of spermidine in Saccharomyces cerevisiae polyamine auxotrophs grown in limiting spermidine. Proc Natl Acad Sci U S A 105:6554-9
Dias, Camila A O; Cano, Veridiana S P; Rangel, Suzana M et al. (2008) Structural modeling and mutational analysis of yeast eukaryotic translation initiation factor 5A reveal new critical residues and reinforce its involvement in protein synthesis. FEBS J 275:1874-88
Cano, Veridiana S P; Jeon, Geoung A; Johansson, Hans E et al. (2008) Mutational analyses of human eIF5A-1--identification of amino acid residues critical for eIF5A activity and hypusine modification. FEBS J 275:44-58
Wolff, E C; Kang, K R; Kim, Y S et al. (2007) Posttranslational synthesis of hypusine: evolutionary progression and specificity of the hypusine modification. Amino Acids 33:341-50
Huang, Yunfei; Higginson, Daniel S; Hester, Lynda et al. (2007) Neuronal growth and survival mediated by eIF5A, a polyamine-modified translation initiation factor. Proc Natl Acad Sci U S A 104:4194-9
Kang, Kee Ryeon; Kim, Yeon Sook; Wolff, Edith C et al. (2007) Specificity of the deoxyhypusine hydroxylase-eukaryotic translation initiation factor (eIF5A) interaction: identification of amino acid residues of the enzyme required for binding of its substrate, deoxyhypusine-containing eIF5A. J Biol Chem 282:8300-8
Park, Myung Hee (2006) The post-translational synthesis of a polyamine-derived amino acid, hypusine, in the eukaryotic translation initiation factor 5A (eIF5A). J Biochem 139:161-9
Park, Jong-Hwan; Aravind, L; Wolff, Edith C et al. (2006) Molecular cloning, expression, and structural prediction of deoxyhypusine hydroxylase: a HEAT-repeat-containing metalloenzyme. Proc Natl Acad Sci U S A 103:51-6
Kim, Yeon Sook; Kang, Kee Ryeon; Wolff, Edith C et al. (2006) Deoxyhypusine hydroxylase is a Fe(II)-dependent, HEAT-repeat enzyme. Identification of amino acid residues critical for Fe(II) binding and catalysis [corrected]. J Biol Chem 281:13217-25
Nishimura, Kazuhiro; Murozumi, Kaori; Shirahata, Akira et al. (2005) Independent roles of eIF5A and polyamines in cell proliferation. Biochem J 385:779-85

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