Mature eukaryotic initiation factor 5A (eIF5A) is the only known protein in eukaryotic cells that contains the unusual amino acid hypusine (N-E-(4-amino-2(R)-hydroxybutyl)lysine). The synthesis of hypusine is essential for the function of eIF5A in eukaryotic cell proliferation and survival. Deoxyhypusine synthase is the first of the two enzymes that catalyzes the maturation of eIF5A. We have subcloned the cDNA encoding bovine and human deoxyhypusine synthase into a pET-11a expression vector, separately. T7-tagged bovine and human deoxyhypusine synthase have been overexpressed in Escherichia coli and purified to homogeneity using T7 antibody affinity chromatography. Activities of the enzyme from both human and bovine have been measured by their ability to convert the eIF5A precursor protein to the intermediate, deoxyhypusine form of eIF5A. Our results have shown that bovine deoxyhypusine synthase has considerably higher activity than human deoxyhypusine synthase in catalyzing the synthesis of deoxyhypusine. (C) 2004 Elsevier Inc. All rights reserved.