The post-translational synthesis of a polyamine-derived amino acid, hypusine, in the eukaryotic translation initiation factor 5A (eIF5A)

Abstract

The eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains the unique polyamine-derived amino acid, hypusine [Nepsilon-(4-amino-2-hydroxybutyl)lysine]. Hypusine is formed in eIF5A by a novel post-translational modification reaction that involves two enzymatic steps. In the first step, deoxyhypusine synthase catalyzes the cleavage of the polyamine spermidine and transfer of its 4-aminobutyl moiety to the epsilon-amino group of one specific lysine residue of the eIF5A precursor to form a deoxyhypusine intermediate. In the second step, deoxyhypusine hydroxylase converts the deoxyhypusine-containing intermediate to the hypusine-containing mature eIF5A. The structure and mechanism of deoxyhypusine synthase have been extensively characterized. Deoxyhypusine hydroxylase is a HEAT-repeat protein with a symmetrical superhelical structure consisting of 8 helical hairpins (HEAT motifs). It is a novel metalloenzyme containing tightly bound iron at the active sites. Four strictly conserved His-Glu pairs were identified as iron coordination sites. The structural fold of deoxyhypusine hydroxylase is entirely different from those of the other known protein hydroxylases such as prolyl 4-hydroxylase and lysyl hydroxylases. The eIF5A protein and deoxyhypusine/hypusine modification are essential for eukaryotic cell proliferation. Thus, hypusine synthesis represents the most specific protein modification known to date, and presents a novel target for intervention in mammalian cell proliferation.

Fig. 1. Scheme of hypusine biosynthesis in eIF5A

The 4-aminobutyl moiety transferred from spermidine to the eIF5A precursor is shaded.

Fig. 2. Comparison of crystal structures of aIF5A and EF-P

A: X-ray structure of archaeal IF5A precursor containing two domains: N-terminal domain (I) and C-terminal domain (II), The location of the lysine residue (in domain I) that undergoes modification to deoxyhypusine or hypusine is indicated. B: Structure of bacterial elongation factor P (EF-P) containing three domains (I–III) in blue, aIF5A in green is superimposed on domains I and II of EF-P. Abbreviations: Hpu, hypusine; Dhp, deoxyhypusine. Panel B reproduced with permission from Ref. 33. Copyright (2004) National Academy of Sciences, USA.

Fig. 3. Mechanism of the deoxyhypusine synthase reaction

The pathways from spermidine leading to deoxyhypusine synthesis and to homospermidine synthesis are indicated by solid arrows. The reversal pathways from eIF5A(Dhp) back to spermidine and from homospermidine to spermidine are indicated by broken arrows. The 4-aminobutyl moiety is shaded and the position of [³H] derived from [1,8-⁸H]spermidine is indicated by *. In the absence of acceptor, the 4-aminobutyl moiety of the enzyme-imine intermediate is cyclized to form Δ¹-pyrroline. The reduction of the transient enzymeimine intermediate to a stable adduct by NaCNBH₃ is indicated by a bold arrow. Abbreviations are: Dap, 1,3-diaminopropane; DHS(Dhp): deoxyhypusine-containing deoxyhypusine synthase. Modified from Ref. 45.

Fig. 4. Tetrameric structure of DHS in complex with NAD (A) and active site of DHS with NAD and GC7 bound (B)

Modified from Ref. 50.

Fig. 5. Sequence and predicted structure of DOHH

A: Sequence alignment of S. cerevisiae and human DOHH protein. A more complete comparison of DOHH sequences is given in Ref. 23. The conserved HE (HisGlu) pairs are boxed. Schematic diagram (B) and 3D model (C) of DOHH in comparison with prolyl 4-hydroxylase (D) Modified from Ref. 23.