Exp Mol Med.  1999 Dec;31(4):210-216.

Characterization of yeast deoxyhypusine synthase: PKC-dependent phosphorylation in vitro and functional domain identification

• Kang KR
• Chung SI

Abstract

The biosynthesis of hypusine [Nepsilon-(4-amino-2-hydroxybutyl)-lysine] occurs in the eIF-5A precursor protein through two step posttranslational modification involving deoxyhypusine synthase which catalyzes transfer of the butylamine moiety of spermidine to the epsilon-amino group of a designated lysine residue and subsequent hydroxylation of this intermediate. This enzyme is exclusively required for cell viability and growth of yeast (Park, M.H. et al., J. Biol. Chem. 273: 1677-1683, 1998). In an effort to understand structure-function relationship of deoxyhypusine synthase, posttranslational modification(s) of the enzyme by protein kinases were carried out for a possible cellular modulation of this enzyme. And also twelve deletion mutants were constructed, expressed in E. coli system, and enzyme activities were examined. The results showed that deoxyhypusine synthase was phosphorylated by PKC in vitro but not by p56lck and p60c-src. Treatment with PMA specifically increased the relative phosphorylation of the enzyme supporting PKC was involved. Phosphoamino acid analysis of this enzyme revealed that deoxyhypusine synthase is mostly phosphorylated on serine residue and weakly on threonine. Removal of Met1-Glu10 (deltaMet1-Glu10) residues from amino terminal showed no effect on the catalytic activity but further deletion (deltaMet1-Ser20) caused loss of enzyme activity. The enzyme with internal deletion, deltaGln197-Asn212 (residues not present in the human enzyme) was found to be inactive. Removal of 5 residues from carboxyl terminal, deltaLys383-Asn387, retained only slight activity. These results suggested that deoxyhypusine synthase is substrate for PKC dependent phosphorylation and requires most of the polypeptide chains for enzyme activity except the first 15 residues of N-terminal despite of N- and C-terminal residues of the enzyme consist of variable regions. Copyright 2000 Academic Press.

Keyword

deoxyhypusine synthase; PKC; phosphorylation; deletion mutation

MeSH Terms

Amine Oxidoreductases/metabolism*
Amine Oxidoreductases/genetics
Amino Acid Motifs
Amino Acid Sequence
Escherichia coli/genetics
Fungal Proteins/metabolism*
Fungal Proteins/genetics
Human
Molecular Sequence Data
NAD/metabolism
Phosphorylation
Promoter Regions (Genetics)
Protein Kinase C/metabolism*
Recombinant Proteins/metabolism
Recombinant Proteins/genetics
Sequence Deletion
Sequence Homology, Amino Acid
Threonine/metabolism
Yeasts/enzymology