Reversal of the deoxyhypusine synthesis reaction. Generation of spermidine or homospermidine from deoxyhypusine by deoxyhypusine synthase

Abstract

Deoxyhypusine synthase catalyzes the first step in hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine) synthesis in a single cellular protein, eIF5A precursor. The synthesis of deoxyhypusine catalyzed by this enzyme involves transfer of the 4-aminobutyl moiety of spermidine to a specific lysine residue in the eIF5A precursor protein to form a deoxyhypusine-containing eIF5A intermediate, eIF5A(Dhp). We recently discovered the efficient reversal of deoxyhypusine synthesis. When eIF5A([3H]Dhp), radiolabeled in the 4-aminobutyl portion of its deoxyhypusine residue, was incubated with human deoxyhypusine synthase, NAD, and 1,3-diaminopropane, [3H]spermidine was formed by a rapid transfer of the radiolabeled 4-aminobutyl side chain of the [3H]deoxyhypusine residue to 1,3-diaminopropane. No reversal was observed with [3H]hypusine protein, suggesting that hydroxylation at the 4-aminobutyl side chain of the deoxyhypusine residue prevents deoxyhypusine synthase-mediated reversal of the modification. Purified human deoxyhypusine synthase also exhibited homospermidine synthesis activity when incubated with spermidine, NAD, and putrescine. Thus it was found that [14C]putrescine can replace eIF5A precursor protein as an acceptor of the 4-aminobutyl moiety of spermidine to form radiolabeled homospermidine. The Km value for putrescine (1.12 mM) as a 4-aminobutyl acceptor, however, is much higher than that for eIF5A precursor (1.5 microM). Using [14C]putrescine as an acceptor, various spermidine analogs were evaluated as donor substrates for human deoxyhypusine synthase. Comparison of spermidine analogs as inhibitors of deoxyhypusine synthesis, as donor substrates for synthesis of deoxyhypusine (or its analog), and for synthesis of homospermidine (or its analog) provides new insights into the intricate specificity of this enzyme and versatility of the deoxyhypusine synthase reaction.