Publications

• Biochemistry and function of hypusine formation on eukaryotic initiation factor 5A.

  Chen K.Y., Liu A.Y.

  Biol Signals 6:105-109(1997) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Deoxyhypusine synthase generates and uses bound NADH in a transient hydride transfer mechanism.

  Wolff E.C., Wolff J., Park M.H.

  Deoxyhypusine is a modified lysine residue. It is formed posttranslationally in the precursor of eukaryotic initiation factor 5A (eIF5A) by deoxyhypusine synthase, employing spermidine as a butylamine donor. In the initial step of this reaction, deoxyhypusine synthase catalyzes the production of N ... >> More

  Deoxyhypusine is a modified lysine residue. It is formed posttranslationally in the precursor of eukaryotic initiation factor 5A (eIF5A) by deoxyhypusine synthase, employing spermidine as a butylamine donor. In the initial step of this reaction, deoxyhypusine synthase catalyzes the production of NADH through dehydrogenation of spermidine. Fluorescence measurements of this reaction revealed a -22-nm blue shift in the emission peak of NADH and a approximately 15-fold increase in peak intensity, characteristics of tightly bound NADH that were not seen by simply mixing NADH and enzyme. The fluorescent properties of the bound NADH can be ascribed to a hydrophobic environment and a rigidly held, open conformation of NADH, features in accord with the known crystal structure of the enzyme. Considerable fluorescence resonance energy transfer from tryptophan 327 in the active site to the dihydronicotinamide ring of NADH was seen. Upon addition of the eIF5A precursor, utilization of the enzyme-bound NADH for reduction of the eIF5A-imine intermediate to deoxyhypusine was reflected by a rapid decrease in the NADH fluorescence, indicating a transient hydride transfer mechanism as an integral part of the reaction. The number of NADH molecules bound approached four/enzyme tetramer; not all of the bound NADH was available for reduction of the eIF5A-imine intermediate. << Less

  J Biol Chem 275:9170-9177(2000) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Cloning and expression of human deoxyhypusine synthase cDNA. Structure-function studies with the recombinant enzyme and mutant proteins.

  Joe Y.A., Wolff E.C., Park M.H.

  Deoxyhypusine synthase catalyzes the first step in the post-translational formation of hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine). cDNA clones encoding deoxyhypusine synthase were isolated from a human HeLa cell library. Full-length cDNA clones encoding a 369-amino acid protein (calculate ... >> More

  Deoxyhypusine synthase catalyzes the first step in the post-translational formation of hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine). cDNA clones encoding deoxyhypusine synthase were isolated from a human HeLa cell library. Full-length cDNA clones encoding a 369-amino acid protein (calculated molecular mass of 40,970 Da) and a shorter cDNA clone that would potentially encode a protein with an internal deletion of 56 amino acids (Asp262-Ser317) were isolated. The deduced amino acid sequence of the human enzyme shows a high degree of identity to that of yeast deoxyhypusine synthase and to the known sequences of tryptic peptides from the rat and Neurospora crassa enzymes. The recombinant enzyme formed upon expression in Escherichia coli effectively catalyzed deoxyhypusine synthesis. Variant human recombinant proteins with (i) a truncation of 48 or 97 NH2-terminal amino acids, (ii) a truncation of 39 COOH-terminal amino acids, or (iii) an internal deletion (Asp262-Ser317) were inactive. A chimeric protein consisting of the complete human sequence and 16 amino acids of the yeast sequence (Gln197-Asn212, not present in the human enzyme) inserted between Glu193 and Gln194 exhibited moderate activity. << Less

  J. Biol. Chem. 270:22386-22392(1995) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Cleavage of spermidine as the first step in deoxyhypusine synthesis. The role of NAD.

  Wolff E.C., Park M.H., Folk J.E.

  The biosynthesis of deoxyhypusine (N-(4-aminobutyl)lysine) occurs by the transfer of the 4-aminobutyl moiety of spermidine to a specific lysine residue in a precursor of eukaryotic translation initiation factor 4D (eIF-4D). Deoxyhypusine synthase, the enzyme that catalyzes this reaction, was purif ... >> More

  The biosynthesis of deoxyhypusine (N-(4-aminobutyl)lysine) occurs by the transfer of the 4-aminobutyl moiety of spermidine to a specific lysine residue in a precursor of eukaryotic translation initiation factor 4D (eIF-4D). Deoxyhypusine synthase, the enzyme that catalyzes this reaction, was purified approximately 700-fold from rat testis. The Km values for the substrates, spermidine, the eIF-4-D precursor protein, and NAD+, were estimated as approximately 1, 0.08, and 30 microM, respectively. After incubation of partially purified enzyme with [1,8-3H]spermidine, NAD+, and the eIF-4D precursor, equal amounts of radioactivity were found in free 1,3-diaminopropane and in protein-bound deoxyhypusine. However, when the protein substrate (eIF-4D precursor) was omitted, radioactivity was found in 1,3-diaminopropane and in delta 1-pyrroline in nearly equal quantities, providing evidence that the cleavage of spermidine occurs, albeit at a slower rate, in the absence of the eIF-4D precursor. That NAD+, which is required for this reaction, functions as the hydrogen acceptor was demonstrated by the fact that radioactivity from spermidine labeled with 3H at position 5 is found in NADH as well as in delta 1-pyrroline. Transfer of this hydrogen from spermidine to the re face of the nicotinamide ring of NAD+, as determined by the use of dehydrogenases of known stereospecificity, defines the first step of deoxyhypusine synthesis as a pro-R, or A, stereospecific dehydrogenation. Based on these findings, an enzyme mechanism involving imine intermediate formation is proposed. << Less

  J Biol Chem 265:4793-4799(1990) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Deoxyhypusine synthase from tobacco. cDNA isolation, characterization, and bacterial expression of an enzyme with extended substrate specificity.

  Ober D., Hartmann T.

  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 th ... >> More

  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. << Less

  J. Biol. Chem. 274:32040-32047(1999) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Enzyme-substrate intermediate formation at lysine 329 of human deoxyhypusine synthase.

  Wolff E.C., Folk J.E., Park M.H.

  Deoxyhypusine (Nepsilon-(4-aminobutyl)lysine) is the key intermediate in the posttranslational synthesis of the unique amino acid, hypusine (Nepsilon-(4-amino-2-hydroxybutyl)lysine). Deoxyhypusine synthase catalyzes the formation of deoxyhypusine by conjugation of the butylamine moiety of spermidi ... >> More

  Deoxyhypusine (Nepsilon-(4-aminobutyl)lysine) is the key intermediate in the posttranslational synthesis of the unique amino acid, hypusine (Nepsilon-(4-amino-2-hydroxybutyl)lysine). Deoxyhypusine synthase catalyzes the formation of deoxyhypusine by conjugation of the butylamine moiety of spermidine to the epsilon-amino group of one specific lysine residue of the eukaryotic translation initiation factor 5A (eIF-5A) precursor protein. However, in the absence of the eIF-5A precursor, catalysis involves only the NAD-dependent cleavage of spermidine to generate 1,3-diaminopropane and a putative 4-carbon amine intermediate that gives rise to Delta1-pyrroline. We have obtained evidence for a covalent enzyme-substrate intermediate that accumulates in the absence of the eIF-5A precursor. Incubation of human recombinant enzyme with [1, 8-3H]spermidine and NAD, followed by reduction with NaBH3CN, resulted in specific radiolabeling of the enzyme. The radioactive component in the reduced enzyme intermediate was identified as deoxyhypusine and was shown to occur at a single locus. The fact that labeled deoxyhypusine was found after treatment with a reducing agent suggests an intermediate with the butylamine moiety derived from spermidine attached through an imine linkage to the epsilon-amino group of a specific lysine residue of the enzyme. This residue has been identified as lysine 329. Separate experiments showing efficient transfer of labeled butylamine moiety from enzyme intermediate to eIF-5A precursor strongly support a reaction mechanism involving an imine intermediate. << Less

  J Biol Chem 272:15865-15871(1997) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Molecular cloning and functional expression of Neurospora deoxyhypusine synthase cDNA and identification of yeast deoxyhypusine synthase cDNA.

  Tao Y., Chen K.Y.

  Deoxyhypusine synthase catalyzes the formation of deoxyhypusine residue on the eIF-5A precursor using spermidine as the substrate. We have purified deoxyhypusine synthase from Neurospora crassa to apparent homogeneity (Tao, Y., and Chen, K. Y. (1995) J. Biol. Chem. 270, 383-386). We have now clone ... >> More

  Deoxyhypusine synthase catalyzes the formation of deoxyhypusine residue on the eIF-5A precursor using spermidine as the substrate. We have purified deoxyhypusine synthase from Neurospora crassa to apparent homogeneity (Tao, Y., and Chen, K. Y. (1995) J. Biol. Chem. 270, 383-386). We have now cloned and characterized the deoxyhypusine synthase cDNA using a reverse genetic approach. Conceptual translation of the nucleotide sequence of the cloned 1258-base pair cDNA revealed an open reading frame containing 353 amino acids with a predicted M(r) of 38,985. The deoxyhypusine synthase cDNA was subcloned into the expression vector pQE60 to produce a 40,000-dalton recombinant protein on SDS-PAGE which exhibited deoxyhypusine synthase activity. A GenBank search showed that the Neurospora deoxyhypusine synthase cDNA possessed significant sequence homology to a previously uncharacterized yeast sequence (accession number U00061 (1994)). The yeast sequence encodes a protein of 387 amino acids that shows 69% of total amino acid identity and 80% of total amino acid similarity to the Neurospora enzyme. Sequence alignment and hydropathy analysis suggest that the yeast sequence represents deoxyhypusine synthase. << Less

  J. Biol. Chem. 270:23984-23987(1995) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Identification of lysine350 of yeast deoxyhypusine synthase as the site of enzyme intermediate formation.

  Wolff E.C., Park M.H.

  The posttranslational formation of deoxyhypusine in the precursor of eukaryotic initiation factor 5A (eIF5A) is catalysed by deoxyhypusine synthase. This NAD-dependent reaction involves transfer of the 4-aminobutyl moiety of spermidine to a single lysine residue in the eIF5A precursor. The present ... >> More

  The posttranslational formation of deoxyhypusine in the precursor of eukaryotic initiation factor 5A (eIF5A) is catalysed by deoxyhypusine synthase. This NAD-dependent reaction involves transfer of the 4-aminobutyl moiety of spermidine to a single lysine residue in the eIF5A precursor. The present study shows evidence for the formation of a covalent enzyme-substrate intermediate between a specific lysine residue (Lys350) of yeast deoxyhypusine synthase and the 4-aminobutyl moiety from spermidine. Substitution of this lysine residue with Arg or Ala totally prevented the formation of the enzyme intermediate and consequently precluded deoxyhypusine synthesis in the eIF5A precursor, leading to the conclusion that the enzyme intermediate formed at Lys350 is critical for deoxyhypusine synthesis activity. The results provide a rational basis for the inability of the mutated deoxyhypusine synthase gene encoding arginine in place of Lys350 to support growth in yeast (Park et al., 1998). The demonstration of the formation of an enzyme-imine intermediate in yeast deoxyhypusine synthase analogous to that of the human enzyme strongly suggest that the enzyme mechanism is conserved in diverse eukaryotes. << Less

  Yeast 15:43-50(1999) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Homospermidine synthase, the first pathway-specific enzyme of pyrrolizidine alkaloid biosynthesis, evolved from deoxyhypusine synthase.

  Ober D., Hartmann T.

  Pyrrolizidine alkaloids are preformed plant defense compounds with sporadic phylogenetic distribution. They are thought to have evolved in response to the selective pressure of herbivory. The first pathway-specific intermediate of these alkaloids is the rare polyamine homospermidine, which is synt ... >> More

  Pyrrolizidine alkaloids are preformed plant defense compounds with sporadic phylogenetic distribution. They are thought to have evolved in response to the selective pressure of herbivory. The first pathway-specific intermediate of these alkaloids is the rare polyamine homospermidine, which is synthesized by homospermidine synthase (HSS). The HSS gene from Senecio vernalis was cloned and shown to be derived from the deoxyhypusine synthase (DHS) gene, which is highly conserved among all eukaryotes and archaebacteria. DHS catalyzes the first step in the activation of translation initiation factor 5A (eIF5A), which is essential for eukaryotic cell proliferation and which acts as a cofactor of the HIV-1 Rev regulatory protein. Sequence comparison provides direct evidence for the evolutionary recruitment of an essential gene of primary metabolism (DHS) for the origin of the committing step (HSS) in the biosynthesis of pyrrolizidine alkaloids. << Less

  Proc. Natl. Acad. Sci. U.S.A. 96:14777-14782(1999) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.