Deoxyhypusine Synthase from Tobacco

Abstract

Deoxyhypusine synthase catalyzes the formation of a deoxyhypusine residue in the translation eukaryotic initiation factor 5A (eIF5A) precursor protein by transferring an aminobutyl moiety from spermidine onto a conserved lysine residue within the eIF5A polypeptide chain. This reaction commences the activation of the initiation factor in fungi and vertebrates. A mechanistically identical reaction is known in the biosynthetic pathway leading to pyrrolizidine alkaloids in plants. Deoxyhypusine synthase from tobacco was cloned and expressed in active form in Escherichia coli. It catalyzes the formation of a deoxyhypusine residue in the tobacco eIF5A substrate as shown by gas chromatography coupled with a mass spectrometer. The enzyme also accepts free putrescine as the aminobutyl acceptor, instead of lysine bound in the eIF5A polypeptide chain, yielding homospermidine. Conversely, it accepts homospermidine instead of spermidine as the aminobutyl donor, whereby the reactions with putrescine and homospermidine proceed at the same rate as those involving the authentic substrates. The conversion of deoxyhypusine synthase-catalyzed eIF5A deoxyhypusinylation pinpoints a function for spermidine in plant metabolism. Furthermore, and quite unexpectedly, the substrate spectrum of deoxyhypusine synthase hints at a biochemical basis behind the sparse and skew occurrence of both homospermidine and its pyrrolizidine derivatives across distantly related plant taxa.The eukaryotic initiation factor 5A (eIF5A), 1 a small 17.4-kDa protein, is activated by a post-translational modification of a specific lysine residue to hypusine (N ⑀ -(4-amino-2-hydroxybutyl)lysine) in an enzyme-catalyzed two-step mechanism (reviewed in Refs. 1 and 2). In the first step, the aminobutyl moiety of the polyamine spermidine is transferred by deoxyhypusine synthase (EC 1.1.1.249) in an NAD ϩ -dependent reaction to the ⑀-amino group of a specific lysine residue in the eIF5A precursor protein to form deoxyhypusine. In the second step, deoxyhypusine hydroxylase (EC 1.14.99.29) catalyzes the hydroxylation of the deoxyhypusine residue to hypusine. Activated eIF5A is the only protein in which the unusual amino acid hypusine has been detected to date (3, 4), the modification is one of the most specific post-translational modifications known (5, 6). eIF5A seems to be ubiquitous among eukaryotes (7) and archaebacteria (8). Its amino acid sequence is highly conserved, and the 12 amino acids surrounding the hypusine residue are identical in all eukaryotes studied. Although known for nearly 2 decades, the function of eIF5A is still obscure. Because of its in vitro activity in stimulating methionyl puromycin synthesis (9), eIF5A was classified as a protein synthesis initiation factor, though subsequent doubts have arisen as to whether initiation of protein synthesis is a major function of this protein (2). Using a yeast mutant, it was shown that depletion of eIF5A causes an immediate inhibition of cell growth but only a moderate inhibition (30%) of total protein synthe...

Evolution and Multifarious Horizontal Transfer of an Alternative Biosynthetic Pathway for the Alternative Polyamine sym-Homospermidine

“…The enzyme deoxyhypusine synthase (DHS) transfers an aminobutyl group from spermidine to a lysine residue in eukaryotic translation initiation factor eIF5A (79) to form the unique residue deoxyhypusine. DHS is also able, as a side reaction, to transfer an aminobutyl group to putrescine to form homospermidine (80). The plant homospermidine synthase, which is used solely for pyrrolizidine alkaloid biosynthesis, is unrelated to HSS and has evolved from DHS by gene duplication and has lost DHS activity but retained the side reaction activity of homospermidine biosynthesis (40,41).…”

Antisense Suppression of Deoxyhypusine Synthase in Tomato Delays Fruit Softening and Alters Growth and Development

“…Evidence supporting this includes the fact that recombinant tomato and tobacco (Nicotiana tabacum) DHS have been shown capable of catalyzing the formation of a deoxyhypusine residue in Arabidopsis (Arabidopsis thaliana) and tobacco eIF-5A substrates, respectively (Ober and Hartmann, 1999;Wang et al, 2001). These observations indicate not only that plant DHS exhibits the same catalytic property as its animal and yeast counterparts (Schwelberger et al, 1993;Park et al, 1997), but also that recombinant plant eIF-5A is capable of being deoxyhypusine modified.…”

“…Full-length cDNA clones encoding DHS and eIF-5A have been isolated from a number of plant species (Chamot and Kuhlemeier, 1992;Ober and Hartmann, 1999;Wang et al, 2001Wang et al, , 2003. Moreover, the recent isolation of DHS from a cDNA expression library prepared from osmotically stressed tomato leaves (Wang et al, 2001) indicates that plant DHS in conjunction with eIF-5A may be involved in facilitating the translation of proteins required for cell death.…”

A New Crystal Structure of Deoxyhypusine Synthase Reveals the Configuration of the Active Enzyme and of an Enzyme·NAD·Inhibitor Ternary Complex

“…DHS is also capable of producing homospermidine from spermidine as a side reaction in the absence of eIF5A(Lys) and presence of putrescine and may be responsible for the presence of homospermidine within cells lacking HSS (39,(41)(42)(43). DHS and HSS employ the same enzymatic mechanism, and the residues defining the active-site tunnel and the NAD binding site are highly conserved.…”