4-aminobutyl Moiety Research Articles

Arginine metabolism regulates human erythroid differentiation through hypusination of eIF5A

AbstractMetabolic programs contribute to hematopoietic stem and progenitor cell (HSPC) fate, but it is not known whether the metabolic regulation of protein synthesis controls HSPC differentiation. Here, we show that SLC7A1/cationic amino acid transporter 1–dependent arginine uptake and its catabolism to the polyamine spermidine control human erythroid specification of HSPCs via the activation of the eukaryotic translation initiation factor 5A (eIF5A). eIF5A activity is dependent on its hypusination, a posttranslational modification resulting from the conjugation of the aminobutyl moiety of spermidine to lysine. Notably, attenuation of hypusine synthesis in erythroid progenitors, by the inhibition of deoxyhypusine synthase, abrogates erythropoiesis but not myeloid cell differentiation. Proteomic profiling reveals mitochondrial translation to be a critical target of hypusinated eIF5A, and accordingly, progenitors with decreased hypusine activity exhibit diminished oxidative phosphorylation. This affected pathway is critical for eIF5A-regulated erythropoiesis, as interventions augmenting mitochondrial function partially rescue human erythropoiesis under conditions of attenuated hypusination. Levels of mitochondrial ribosomal proteins (RPs) were especially sensitive to the loss of hypusine, and we find that the ineffective erythropoiesis linked to haploinsufficiency of RPS14 in chromosome 5q deletions in myelodysplastic syndrome is associated with a diminished pool of hypusinated eIF5A. Moreover, patients with RPL11-haploinsufficient Diamond-Blackfan anemia as well as CD34+ progenitors with downregulated RPL11 exhibit a markedly decreased hypusination in erythroid progenitors, concomitant with a loss of mitochondrial metabolism. Thus, eIF5A-dependent protein synthesis regulates human erythropoiesis, and our data reveal a novel role for RPs in controlling eIF5A hypusination in HSPCs, synchronizing mitochondrial metabolism with erythroid differentiation.

Role of Polyamines and Hypusine in β Cells and Diabetes Pathogenesis.

The polyamines—putrescine, spermidine, and spermine—are polycationic, low molecular weight amines with cellular functions primarily related to mRNA translation and cell proliferation. Polyamines partly exert their effects via the hypusine pathway, wherein the polyamine spermidine provides the aminobutyl moiety to allow posttranslational modification of the translation factor eIF5A with the rare amino acid hypusine (hydroxy putrescine lysine). The “hypusinated” eIF5A (eIF5Ahyp) is considered to be the active form of the translation factor necessary for the translation of mRNAs associated with stress and inflammation. Recently, it has been demonstrated that activity of the polyamines-hypusine circuit in insulin-producing islet β cells contributes to diabetes pathogenesis under conditions of inflammation. Elevated levels of polyamines are reported in both exocrine and endocrine cells of the pancreas, which may contribute to endoplasmic reticulum stress, oxidative stress, inflammatory response, and autophagy. In this review, we have summarized the existing research on polyamine-hypusine metabolism in the context of β-cell function and diabetes pathogenesis.

Post-translational formation of hypusine in eIF5A: implications in human neurodevelopment

Hypusine [Nε-(4-amino-2-hydroxybutyl)lysine] is a derivative of lysine that is formed post-translationally in the eukaryotic initiation factor 5A (eIF5A). Its occurrence at a single site in one cellular protein defines hypusine synthesis as one of the most specific post-translational modifications. Synthesis of hypusine involves two enzymatic steps: first, deoxyhypusine synthase (DHPS) cleaves the 4-aminobutyl moiety of spermidine and transfers it to the ε-amino group of a specific lysine residue of the eIF5A precursor protein to form an intermediate, deoxyhypusine [Nε-(4-aminobutyl)lysine]. This intermediate is subsequently hydroxylated by deoxyhypusine hydroxylase (DOHH) to form hypusine in eIF5A. eIF5A, DHPS, and DOHH are highly conserved in all eukaryotes, and both enzymes exhibit a strict specificity toward eIF5A substrates. eIF5A promotes translation elongation globally by alleviating ribosome stalling and it also facilitates translation termination. Hypusine is required for the activity of eIF5A, mammalian cell proliferation, and animal development. Homozygous knockout of any of the three genes, Eif5a, Dhps, or Dohh, leads to embryonic lethality in mice. eIF5A has been implicated in various human pathological conditions. A recent genetic study reveals that heterozygous germline EIF5A variants cause Faundes–Banka syndrome, a craniofacial–neurodevelopmental malformations in humans. Biallelic variants of DHPS were identified as the genetic basis underlying a rare inherited neurodevelopmental disorder. Furthermore, biallelic DOHH variants also appear to be associated with neurodevelopmental disorder. The clinical phenotypes of these patients include intellectual disability, developmental delay, seizures, microcephaly, growth impairment, and/or facial dysmorphisms. Taken together, these findings underscore the importance of eIF5A and the hypusine modification pathway in neurodevelopment in humans.

Development of an activity assay for characterizing deoxyhypusine synthase and its diverse reaction products.

Deoxyhypusine synthase transfers an aminobutyl moiety from spermidine to the eukaryotic translation initiation factor 5A (eIF5A) in the first step of eIF5A activation. This exclusive post-translational modification is conserved in all eukaryotes. Activated eIF5A has been shown to be essential for cell proliferation and viability. Recent reports have linked the activation of eIF5A to several human diseases. Deoxyhypusine synthase, which is encoded by a single gene copy in most eukaryotes, was duplicated in several plant lineages during evolution, the copies being repeatedly recruited to pyrrolizidine alkaloid biosynthesis. However, the function of many of these duplicates is unknown. Notably, deoxyhypusine synthase is highly promiscuous and can catalyze various reactions, often of unknown biological relevance. To facilitate in-depth biochemical studies of this enzyme, we report here the development of a simple and robust invitro enzyme assay. It involves precolumn derivatization of the polyamines taking part in the reaction and avoids the need for the previously used radioactively labeled tracers. The derivatized polyamines are quantified after high-performance liquid chromatography coupled to diode array and fluorescence detectors. By performing kinetic analyses of deoxyhypusine synthase and its paralog from the pyrrolizidine alkaloid-producing plant Seneciovernalis, we demonstrate that the assay unequivocally differentiates the paralogous enzymes. Furthermore, it detects and quantifies, in a single assay, the side reactions that occur in parallel to the main reaction. The presented assay thus provides a detailed biochemical characterization of deoxyhypusine synthase and its paralogs.

Half Way to Hypusine-Structural Basis for Substrate Recognition by Human Deoxyhypusine Synthase.

Deoxyhypusine synthase (DHS) is a transferase enabling the formation of deoxyhypusine, which is the first, rate-limiting step of a unique post-translational modification: hypusination. DHS catalyses the transfer of a 4-aminobutyl moiety of polyamine spermidine to a specific lysine of eukaryotic translation factor 5A (eIF5A) precursor in a nicotinamide adenine dinucleotide (NAD)-dependent manner. This modification occurs exclusively on one protein, eIF5A, and it is essential for cell proliferation. Malfunctions of the hypusination pathway, including those caused by mutations within the DHS encoding gene, are associated with conditions such as cancer or neurodegeneration. Here, we present a series of high-resolution crystal structures of human DHS. Structures were determined as the apoprotein, as well as ligand-bound states at high-resolutions ranging from 1.41 to 1.69 Å. By solving DHS in complex with its natural substrate spermidine (SPD), we identified the mode of substrate recognition. We also observed that other polyamines, namely spermine (SPM) and putrescine, bind DHS in a similar manner as SPD. Moreover, we performed activity assays showing that SPM could to some extent serve as an alternative DHS substrate. In contrast to previous studies, we demonstrate that no conformational changes occur in the DHS structure upon spermidine-binding. By combining mutagenesis and a light-scattering approach, we show that a conserved “ball-and-chain” motif is indispensable to assembling a functional DHS tetramer. Our study substantially advances our knowledge of the substrate recognition mechanism by DHS and may aid the design of pharmacological compounds for potential applications in cancer therapy.

Discovery of niclosamide and its O-alkylamino-tethered derivatives as potent antibacterial agents against carbapenemase-producing and/or colistin resistant Enterobacteriaceae isolates

Carbapenemase-producing Enterobacteriaceae (CPE) represents the most worrisome evolution of the antibiotic resistance crisis, which is almost resistant to most of available antibiotics. This situation is getting even worse particularly due to the recent emergence of colistin resistance. Herein, niclosamide, an FDA-approved traditional drug, and its novel O-alkylamino-tethered derivatives were discovered as new and potent antibacterial agents against carbapenemase-producing and/or colistin resistant Enterobacteriaceae isolates. Among these molecules, compound 10 (HJC0431) with 4-aminobutyl moiety showed the broad antibacterial activities, effective against 6 strains. In vitro checkerboard and time-kill course studies demonstrated the synergistic effects of the screened compounds with colistin against the corresponding strains with various degrees.

Relevance of the Axis Spermidine/eIF5A for Plant Growth and Development.

One key role of the essential polyamine spermidine in eukaryotes is to provide the 4-aminobutyl moiety group destined to the post-translational modification of a lysine in the highly conserved translation factor eIF5A. This modification is catalyzed by two sequential enzymatic steps leading to the activation of eIF5A by the conversion of one conserved lysine to the unusual amino acid hypusine. The active translation factor facilitates the sequence-specific translation of polyproline sequences that otherwise cause ribosome stalling. In spite of the well-characterized involvement of active eIF5A in the translation of proline repeat-rich proteins, its biological role has been recently elucidated only in mammals, and it is poorly described at the functional level in plants. Here we describe the alterations in plant growth and development caused by RNAi-mediated conditional genetic inactivation of the hypusination pathway in Arabidopsis thaliana by knocking-down the enzyme deoxyhypusine synthase. We have uncovered that spermidine-mediated activation of eIF5A by hypusination is involved in several aspects of plant biology such as the control of flowering time, the aerial and root architecture, and root hair growth. In addition this pathway is required for adaptation to challenging growth conditions such as high salt and high glucose medium and to elevated concentrations of the plant hormone ABA. We have also performed a bioinformatic analysis of polyproline-rich containing proteins as putative eIF5A targets to uncover their organization in clusters of protein networks to find molecular culprits for the disclosed phenotypes. This study represents a first attempt to provide a holistic view of the biological relevance of the spermidine-dependent hypusination pathway for plant growth and development.

Deciphering the Translation Initiation Factor 5A Modification Pathway in Halophilic Archaea.

Translation initiation factor 5A (IF5A) is essential and highly conserved in Eukarya (eIF5A) and Archaea (aIF5A). The activity of IF5A requires hypusine, a posttranslational modification synthesized in Eukarya from the polyamine precursor spermidine. Intracellular polyamine analyses revealed that agmatine and cadaverine were the main polyamines produced in Haloferax volcanii in minimal medium, raising the question of how hypusine is synthesized in this halophilic Archaea. Metabolic reconstruction led to a tentative picture of polyamine metabolism and aIF5A modification in Hfx. volcanii that was experimentally tested. Analysis of aIF5A from Hfx. volcanii by LC-MS/MS revealed it was exclusively deoxyhypusinylated. Genetic studies confirmed the role of the predicted arginine decarboxylase gene (HVO_1958) in agmatine synthesis. The agmatinase-like gene (HVO_2299) was found to be essential, consistent with a role in aIF5A modification predicted by physical clustering evidence. Recombinant deoxyhypusine synthase (DHS) from S. cerevisiae was shown to transfer 4-aminobutyl moiety from spermidine to aIF5A from Hfx. volcanii in vitro. However, at least under conditions tested, this transfer was not observed with the Hfx. volcanii DHS. Furthermore, the growth of Hfx. volcanii was not inhibited by the classical DHS inhibitor GC7. We propose a model of deoxyhypusine synthesis in Hfx. volcanii that differs from the canonical eukaryotic pathway, paving the way for further studies.

Essential role of eIF5A-1 and deoxyhypusine synthase in mouse embryonic development

The eukaryotic initiation factor 5A (eIF5A) contains a polyamine-derived amino acid, hypusine [N(ε)-(4-amino-2-hydroxybutyl)lysine]. Hypusine is formed post-translationally by the addition of the 4-aminobutyl moiety from the polyamine spermidine to a specific lysine residue, catalyzed by deoxyhypusine synthase (DHPS), and subsequent hydroxylation by deoxyhypusine hydroxylase (DOHH). The eIF5A precursor protein and both of its modifying enzymes are highly conserved, suggesting a vital cellular function for eIF5A and its hypusine modification. To address the functions of eIF5A and the first modification enzyme, DHPS, in mammalian development, we knocked out the Eif5a or the Dhps gene in mice. Eif5a heterozygous knockout mice and Dhps heterozygous knockout mice were viable and fertile. However, homozygous Eif5a1 (gt/gt) embryos and Dhps (gt/gt) embryos died early in embryonic development, between E3.5 and E7.5. Upon transfer to in vitro culture, homozygous Eif5a (gt/gt) or Dhps (gt/gt) blastocysts at E3.5 showed growth defects when compared to heterozygous or wild type blastocysts. Thus, the knockout of either the eIF5A-1 gene (Eif5a) or of the deoxyhypusine synthase gene (Dhps) caused early embryonic lethality in mice, indicating the essential nature of both eIF5A-1 and deoxyhypusine synthase in mammalian development.

Synthesis of a psilocin hapten and a protein-hapten conjugate.

Derivatives of psilocin with omega-functionalized alkyl spacers in position 1 of the indole ring were synthesized as haptens for use in a radioimmunoassay. Whereas the psilocin analogues with a 3-aminopropyl and a 4-aminobutyl moiety at the indole nitrogen decomposed during synthesis, the analogous 3-carboxypropyl psilocin derivative proved to be stable. This compound was coupled to bovine serum albumin (BSA) using the N-hydroxysuccinimide ester-mediated conjugation. The protein-hapten conjugate was characterized by matrix-assisted laser desorption ionization mass spectrometry. The mass spectrometry data indicated an average incorporation ratio of 4-5 molecules of psilocin hapten per molecule of BSA.

Functional significance of eIF5A and its hypusine modification in eukaryotes

The unusual basic amino acid, hypusine [N(epsilon)-(4-amino-2-hydroxybutyl)-lysine], is a modified lysine with the addition of the 4-aminobutyl moiety from the polyamine spermidine. This naturally occurring amino acid is a product of a unique posttranslational modification that occurs in only one cellular protein, eukaryotic translation initiation factor 5A (eIF5A, eIF-5A). Hypusine is synthesized exclusively in this protein by two sequential enzymatic steps involving deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). The deoxyhypusine/hypusine synthetic pathway has evolved in archaea and eukaryotes, and eIF5A, DHS and DOHH are highly conserved suggesting a vital cellular function of eIF5A. Gene disruption and mutation studies in yeast and higher eukaryotes have provided valuable information on the essential nature of eIF5A and the deoxyhypusine/hypusine modification in cell growth and in protein synthesis. In view of the extraordinary specificity and functional significance of hypusine-containing eIF5A in mammalian cell proliferation, eIF5A and the hypusine biosynthetic enzymes are novel potential targets for intervention in aberrant cell proliferation.

Piperidones with activity against Plasmodium falciparum

The increasing resistance of the malaria parasites has enforced new strategies of finding new drug targets. We have isolated two genes involved in spermidine metabolism, encoding deoxyhypusine synthase (DHS) and eukaryotic initiation factor 5A (eIF-5A) in the malaria parasites. eIF-5A is activated by the formation of the unusual amino acid hypusine. This process occurs in two steps. DHS transfers an aminobutyl moiety from the triamine spermidine to a specific lysine residue in the eIF-5A precursor protein to form deoxyhypusine. In a second step, deoxyhypusine hydroxylase (DHH), completes hypusine biosynthesis. We used DHH inhibitors, being effective in mammalian cells, to study an antiplasmodicidal effect in Plasmodium falciparum. Experiments with the antifungal drug ciclopiroxolamine, an alpha-hydroxypyridone, and the plant amino acid L: -mimosine, a 4-pyridone, resulted in an antiplasmodial effect in vitro. Using mimosine as a lead structure, alkyl 4-oxo-piperidine 3-carboxylates were found to have the most efficient antiplasmodial effects in vitro and in vivo.

The Post-Translational Synthesis of a Polyamine-Derived Amino Acid, Hypusine, in the Eukaryotic Translation Initiation Factor 5A (eIF5A)

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.

Translation initiation factor eIF-5A from Plasmodium falciparum.

Eukaryotic translation initiation factor (eIF-5A) is a highly conserved and essential protein that contains the unique amino acid hypusine. The first step in the post-translational biosynthesis of hypusine, the transfer of an aminobutyl moiety from the polyamine substrate spermidine to the -amino group of a specific lysine residue in the eIF-5A precursor, is catalyzed by the enzyme deoxyhypusine synthase. A cDNA encoding a protein homologous to eIF-5A was isolated by plaque hybridization from a cDNA library of Plasmodium falciparum. The cloned cDNA contains an open reading frame encoding a protein of 161 amino acids, which shares a high sequence identity with other eukaryotic eIF-5A sequences. A phylogenetic tree constructed with eIF-5A from P. falciparum and 16 other eIF-5A sequences of eukaryotic and archaeal origin reveals that plasmodial eIF-5A together with other apicomplexan eIF-5A show a higher degree of homology to plant proteins than to animal and fungal sequences. The plasmodial eIF-5A gene was expressed as a six-histidine tagged fusion protein in Escherichia coli. Radioactive incorporation studies with [1,8-3H] spermidine indicated that this protein can serve as a substrate for human deoxyhypusine synthase. Results of quantitative real-time PCR studies with synchronized erythrocytic stages of P. falciparum revealed no significant induction or downregulation but only some variation in the expression level of plasmodial eIF-5A in ring, trophozoite and schizont stage.

Reversal of the Deoxyhypusine Synthesis Reaction: GENERATION OF SPERMIDINE OR HOMOSPERMIDINE FROM DEOXYHYPUSINE BY DEOXYHYPUSINE SYNTHASE

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.

Molecular Evolution by Change of Function: ALKALOID-SPECIFIC HOMOSPERMIDINE SYNTHASE RETAINED ALL PROPERTIES OF DEOXYHYPUSINE SYNTHASE EXCEPT BINDING THE eIF5A PRECURSOR PROTEIN

Deoxyhypusine synthase participates in the post-translational activation of the eukaryotic initiation factor 5A (eIF5A). The enzyme transfers the aminobutyl moiety of spermidine to a specific lysine residue in the eIF5A precursor protein, i.e. eIF5A(lys). Homospermidine synthase catalyzes an analogous reaction but uses putrescine instead of eIF5A(lys) as substrate yielding the rare polyamine homospermidine as product. Homospermidine is an essential precursor in the biosynthesis of pyrrolizidine alkaloids, an important class of plant defense compounds against herbivores. Sequence comparisons of the two enzymes indicate an evolutionary origin of homospermidine synthase from ubiquitous deoxyhypusine synthase. The two recombinant enzymes from Senecio vernalis were purified, and their properties were compared. Protein-protein binding and kinetic substrate competition studies confirmed that homospermidine synthase, in comparison to deoxyhypusine synthase, lost the ability to bind the eIF5A(lys) to its surface. The two enzymes show the same unique substrate specificities, catalyze the aminobutylation of putrescine with the same specific activities, and exhibit almost identical Michaelis kinetics. In conclusion, homospermidine synthase behaves like a deoxyhypusine synthase that lost its major function (aminobutylation of eIF5A precursor protein) but retained unaltered its side activity (aminobutylation of putrescine). It is suggested as having evolved from deoxyhypusine synthase by gene duplication and being recruited for a new function.

Transcript levels of the eukaryotic translation initiation factor 5A gene peak at early G(1) phase of the cell cycle in the dinoflagellate Crypthecodinium cohnii.

A cDNA encoding a eukaryotic translation initiation factor 5A (eIF-5A) homolog in heterotrophic dinoflagellate Crypthecodinium cohnii (CceIF-5A) was isolated through random sequencing of a cDNA library. The predicted amino acid sequence possesses the 12 strictly conserved amino acids around lysine 52 (equivalent to lysine 50 or 51 in other eukaryotes). A single 1.2-kb band was detected in Northern blot analysis. In synchronized C. cohnii cells, the transcript level peaked at early G(1) and decreased dramatically on the entry to S phase. Although this has not been previously reported, studies of budding yeast (Saccharomyces cerevisiae) and certain mammalian cell types suggest a role for eIF-5A in the G(1)/S transition of the eukaryotic cell cycle. Phylogenetic trees constructed with 26 other published eIF-5A sequences suggest that CceIF-5A, while falling within the eukaryotic branches, forms a lineage separate from those of the plants, animals, and archaebacteria. The posttranslational modification of eIF-5A by a transfer of a 4-aminobutyl moiety from spermidine to conserved lysine 50 or 51, forming amino acid hypusine, is the only demonstrated specific function of polyamines in cell proliferation. It has been suggested that polyamines stimulate population growth of bloom-forming dinoflagellates in the sea. We demonstrate here putrescine-stimulated cell proliferation. Furthermore, ornithine decarboxylase inhibitor D-difluoromethylornithine and the specific hypusination inhibitor N-guanyl-1,7-diaminoheptane exhibited inhibitory effects in two species of dinoflagellates. The possible links of polyamines and saxitoxin synthesis to the arginine cycle are also discussed.

Deoxyhypusine Synthase from Tobacco: cDNA ISOLATION, CHARACTERIZATION, AND BACTERIAL EXPRESSION OF AN ENZYME WITH EXTENDED SUBSTRATE SPECIFICITY

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.

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

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 suggests that the enzyme mechanism is conserved in diverse eukaryotes. Copyright © 1999 John Wiley & Sons, Ltd.

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

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.