Homospermidine Synthase Research Articles

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.

Evidence for general occurrence of homospermidine in plants and its supposed origin as by-product of deoxyhypusine synthase

Deoxyhypusine synthase (DHS) is involved in the post-translational activation of the eukaryotic initiation factor 5A (eIF5A) and, as a side-reaction, catalyzes the formation of homospermidine if its substrate, the eIF5A precursor protein, is replaced by putrescine. Plant homospermidine synthase is assumed to be phylogenetically derived from DHS; it represents a DHS having lost its intrinsic activity. The enzyme is expressed in plants producing pyrrolizidine alkaloids where it catalyzes the formation of homospermidine the unique precursor of pyrrolizidine alkaloids. Here we show that 29 species randomly selected from 18 angiosperm families as well as a few other terrestrial plant species, all were able to produce small amounts of homospermidine. Basing on these results and in the context of literature on the occurrence of homospermidine in the organismic kingdoms, a universal occurrence of homospermidine is assumed and ubiquitous DHS is suggested to be responsible for its formation. The synthesis of homospermidine as an enzymatic by-product of an essential enzyme is discussed in respect to the evolutionary origin of homospermidine synthase and the biosynthetic pathway of pyrrolizidine alkaloids.

Heterologous expression of a bacterial homospermidine synthase gene in transgenic tobacco: effects on the polyamine pathway.

Homospermidine synthase (HSS) is a branch-point enzyme that links the secondary pathway (pyrrolizidine alkaloids) to primary metabolism (polyamines). Since the diamine putrescine is a precursor of homospermidine and nicotine in tobacco, we performed heterologous expression of a bacterial homospermidine synthase gene (hss)in Nicotiana tabacum and determined the effect on free and conjugated polyamine levels. The hss gene from Rhodopseudomonas viridis was placed under the control of the cauliflower mosaic virus (CaMV) 35S promoter in Agrobacterium tumefaciens Ti-plasmid in sense and antisense orientation and both hss constructs were transformed into tobacco plants. Expression of the hss gene was verified by "Northern" and "Southern Blot" analysis. 2 transgenic sense lines were generated from 1000 calli which showed weak expression of homospermidine synthase, i.e. 50 pktal/mg protein and 45 pktal/mg protein. These transgenic sense plants showed a significantly decreased content of free spermidine while the pool of conjugated spermidine was not affected. The 2 sense plants exhibited a range of abnormal phenotypes such as dwarfness and stunted growth. Homospermidine was sporadically detectable in wild type tobacco. To our knowledge, this is the first biotechnological approach to express a prokaryotic homospermidine synthase gene in tobacco plants.

Effect of drugs inhibiting spermidine biosynthesis and metabolism on the in vitro development of Plasmodium falciparum.

Treatment of Plasmodium falciparum with the potent inhibitor dicyclohexylamine completely arrests in vitro cell proliferation of the chloroquine-susceptible P. falciparum strain NF54 and the R strain, which shows less sensivity to chloroquine. The average inhibitory concentration (IC50) values determined for both strains revealed different inhibition profiles. The IC50 value for the chloroquine-sensitive NF54 strain was 97 microM and 501 microM for the R strain. Monitoring polyamine pools after treatment with dicyclohexylamine leads to a significant decrease in the intracellular spermidine content, which was nearly reversed by supplementation with spermidine. Since spermidine is an important precursor for the biosynthesis of hypusine and homospermidine in eukaryotes, we studied the developmental effect on both P. falciparum strains of 1,7-diaminoheptane as an inhibitor of deoxyhypusine synthase (EC 1.1.1.249) in mammalian cells, and agmatine as a moderate inhibitor of homospermidine synthase (EC 2.5.1.44). Inhibition profiles with 1,7-diaminoheptane resulted in an IC50 value of 466 microM for the NF54 strain and 319 microM for the R strain. Spermidine pools changed significantly. Inhibition with agmatine caused a strong decrease in parasitemia for the chloroquine-susceptible NF54 strain, with a determined IC50 value of 431 microM and an IC50 value of 340 microM for the less chloroquine-susceptible R strain. Spermidine was not detectable after inhibition. The uncommon triamine homospermidine occurred in both P. falciparum strains. To our knowledge this is the first evidence of homospermidine in P. falciparum. The use of specific inhibitors of spermidine metabolism might be a novel strategy for the design of new antimalarials, and suggests the occurrence of both enzymes in the parasite.

Cloning and expression of homospermidine synthase from Senecio vulgaris: a revision

Homospermidine synthase, which catalyses the first pathway-specific reaction in pyrrolizidine alkaloid biosynthesis, was cloned from root cultures of Senecio vulgaris and expressed in E. coli. The open reading frame encodes a protein of 370 amino acids with a molecular mass of 40,740 Da. The enzyme is strictly dependent on spermidine as aminobutyl donor since it cannot be substituted by putrescine. The homospermidine synthase from S. vulgaris showed 97.9 and 99.3% nucleic acid identity with two HSS sequences from the closely related species Senecio vernalis. This report also revises data from a previous publication (Kaiser, A., 1999. Cloning and expression of a cDNA encoding homospermidine synthase from Senecio vulgaris (Asteraceae) in Escherichia coli. Plant J. 19, 195–201.) that is incorrect.

Phylogenetic origin of a secondary pathway: the case of pyrrolizidine alkaloids.

Recent studies have revealed high sequence similarity between homospermidine synthase (HSS), the first pathway-specific enzyme in the biosynthesis of pyrrolizidine alkaloids, a class of sporadically occurring plant defence compounds, and deoxyhypusine synthase (DHS), a ubiquitous enzyme involved in the post-translational activation of the eukaryotic initiation factor 5A (eIF5A). The recruitment of DHS during the evolution of the alkaloid pathway is discussed and interpreted as evolution by change of function.

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

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.

Chlorella Virus PBCV-1 Encodes a Functional Homospermidine Synthase

Sequence analysis of the 330-kb genome of chlorella virus Paramecium bursaria chlorella virus 1 (PBCV-1) revealed an open reading frame, A237R, that encodes a protein with 34% amino acid identity to homospermidine synthase from Rhodopseudomonas viridis. Expression of the a237r gene product in Escherichia coli established that the recombinant enzyme catalyzes the NAD+-dependent formation of homospermidine from two molecules of putrescine. The a237r gene is expressed late in PBCV-1 infection. Both uninfected and PBCV-1-infected chlorella, as well as PBCV-1 virions, contain homospermidine, along with the more common polyamines putrescine, spermidine, and cadaverine. The total number of polyamine molecules per virion (∼539) is too small to significantly neutralize the virus double-stranded DNA (>660,000 nucleotides). Consequently, the biological significance of the homospermidine synthase gene is unknown. However, the gene is widespread among the chlorella viruses. To our knowledge, this is the first report of a virus encoding an enzyme involved in polyamine biosynthesis.

Cloning and expression of a cDNA encoding homospermidine synthase from Senecio vulgaris (Asteraceae) in Escherichia coli.

The enzyme homospermidine synthase catalyzes the NAD+-dependent conversion of 2 mol putrescine into homospermidine. Instead of putrescine, spermidine can substitute for the first putrescine moiety in plants, in which case diaminopropane instead of ammonia is released. The enzyme facilitates the formation of the 'uncommon' polyamine homospermidine which is an important precursor in the biosynthesis of pyrrolizidine alkaloids. The first plant homospermidine synthase was purified to apparent chemical homogenity from the root tissue culture Senecio vernalis (Asteraceae) (Böttcher et al. 1994, Can. J. Chem. 72, 80-85; Ober 1997, Dissertation). Four endopeptidase LysC fragments were sequenced from the purified protein. With the aid of degenerate primers against these peptides, a cDNA encoding homospermidine synthase was now cloned and characterized from Senecio vulgaris. The nucleotide sequence of the cloned cDNA revealed an open reading frame of 1155-base pairs containing 385 amino acids with a predicted Mr of 44500. GenBank research revealed that the deduced amino acid sequence shows 59% identity to human deoxyhypusine synthase. The homospermidine synthase encoding cDNA was subcloned into the expression vector pet15b and overexpressed in E. coli. The recombinant enzyme formed upon expression catalyzed homospermidine synthesis.

Retarded growth of an Escherichia coli mutant deficient in spermidine synthase can be unspecifically repaired by addition of various polyamines

The Escherichia coli mutant speE deficient in the gene encoding for spermidine synthase has no absolute requirement for spermidine but shows a retarded growth rate. This growth retardation could be unspecifically restored to the respective wild type level by exogenously supplied polyamines such as spermidine, spermine and homospermidine as well as the diamines putrescine and cadaverine. In comparison to the respective wild type, the mutant shows a two-fold increased level of endogenous putrescine but displays a reduced ability to accumulate the diamines putrescine and cadaverine. The ability to accumulate polyamines is not affected. The deleted spermidine synthase gene of the mutant was substituted by heterologous expression of the hss gene from Rhodopseudomonas viridis encoding homospermidine synthase.

Biosynthetic incorporation of the aminobutyl group of spermidine into pyrrolizidine alkaloids

In short-term tracer experiments [ 3H]putrescine and [ 14C]spermidine were fed simultaneously to root cultures of Senecio vulgaris. The specific incorporation of the two tracers into putrescine, spermidine and pyrrolizidine alkaloids was followed over a period of 30 to 480 min. The results show that the aminobutyl moiety of spermidine is directly incorporated into the necine base of pyrrolizidine alkaloids. The same experiment carried out in the presence of the diamine oxidase inhibitor β-hydroxyethylhydrazine, which blocks the alkaloid biosynthesis after homospermidine, revealed the same specific incorporation into accumulating homospermidine. The results are in accordance with the substrate specificity of plant homospermidine synthase (EC 2.5.1.44) which uses both putrescine and spermidine as donor for the amino butyl group. Under physiological conditions more than half of the aminobutyl moiety of homospermidine comes directly from spermidine. This is the first report for the role of spermidine as aminobutyl donor in alkaloid biosynthesis. The importance of the linkage between spermidine and pyrrolizidine alkaloid biosynthesis is discussed. © 1997 Elsevier Science Ltd. All rights reserved

Purification, molecular cloning and expression in Escherichia coli of homospermidine synthase from Rhodopseudomonas viridis.

Homospermidine synthase (HSS) catalyzes the synthesis of the polyamine homospermidine from 2 mol putrescine in an NAD(+)-dependent reaction. In this study, the enzyme was purified from anaerobically grown cultures of the photosynthetic bacterium Rhodopseudomonas viridis to electrophoretic homogeneity using a three-step procedure. The enzyme was shown to be a homodimer of 52-kDa subunits. Six endopeptidase LysC fragments were sequenced from the purified protein. With the aid of degenerate primers designed against these peptides, specific PCR products from R. viridis DNA were obtained that were used as hybridization probes to isolate the hss gene from a library constructed in lambda EMBL4. The hss gene and flanking regions were sequenced and were shown to exist as a single copy in the R. viridis genome. HSS is translated from a monocistronic mRNA and possesses no detectable similarity to previously sequenced gene products. Escherichia coli, which lacks HSS activity, was transformed with an expression plasmid containing the hss coding region under the control of a bacteriophage T7 promoter. Upon induction, transformed F. coli cells accumulate enzymatically active and highly stable R. viridis HSS at levels corresponding to 40-50% of the soluble protein in crude extracts.

Homospermidine synthase of Rhodopseudomonas viridis: Substrate specificity and effects of the heterologously expressed enzyme on polyamine metabolism of Escherichia coli.

Homospermidine synthase (HSS) catalyses the formation of the polyamine homospermidine from 2 mol of putrescine. The general and kinetic properties of purified HSS from Rhodopseudomonas viridis are given and compared with those of the respective enzymes from other sources. The R. viridis enzyme is shown to catalyse a number of side reactions: (I) In the presence of putrescine or spermidine as donors of the 4-aminobutyl moiety, various homologous diamines are transformed into the respective N-(4-aminobutyl)derivatives. (II) In the absence of putrescine, spermidine as a substrate yields homospermidine, putrescine and diaminopropane as reaction products. The mechanism of the reactions catalysed by HSS and its role in the formation of uncommon bacterial polyamines are discussed. Overexpression of the homospermidine synthase (hss) gene in Escherichia coli revealed the formation of two HSS-products, homospermidine and N-(4-aminobutyl)-cadaverine, which are absent from wild-type E. coli. Expression of the hss gene in E. coli does not dramatically affect the pool concentrations of the cellular polyamines.

A New Polyamine 4-Aminobutylcadaverine: OCCURRENCE AND ITS BIOSYNTHESIS IN ROOT NODULES OF ADZUKI BEAN PLANT VIGNA ANGULARIS

Root nodules of adzuki bean plant (Vigna angularis) contained a novel polyamine. The chemical structure of the new polyamine was determined to be NH2(CH2)5-NH(CH2)4NH2 (4-aminobutylcadaverine) based on gas chromatography-mass spectrometry. The occurrence of 4-aminobutylcadaverine was specific to the root nodules, since the unusual triamine was not detected in other organs of the adzuki bean plant. Bacteroids, isolated from root nodules, contained both sym-homospermidine and 4-aminobutylcadaverine, whereas the plant cytosol fraction contained large quantities of putrescine and cadaverine. A cell-free extract of bacteroids showed the ability to form this triamine from putrescine and cadaverine under the presence of NAD+ and K+. 1,3-Diaminopropane and NADH were inhibitory for the synthesis of both sym-homospermidine and 4-aminobutylcadaverine. [1,4-15N]Putrescine was incorporated not only into sym-homospermidine but also into 4-aminobutylcadaverine by the cell-free extract of bacteroids when incubated with excess cadaverine. Analysis of the fragment ion peaks in the 15N-enriched 4-aminobutylcadaverine indicated the transfer of a aminobutyl moiety to the amino terminus of cadaverine. These results suggest that, in adzuki bean, 4-aminobutylcadaverine is formed through the action of homospermidine synthase in nodule bacteroids under a cadaverine-rich environment.

Biosynthesis of pyrrolizidine alkaloids: putrescine and spermidine are essential substrates of enzymatic homospermidine formation

sym-Homospermidine, the first pathway-specific intermediate of pyrrolizidine alkaloid (PA) biosynthesis, is formed from two moles of putrescine in an NAD+-dependent reaction catalyzed by homospermidine synthase (HSS). In the overall process catalyzed by HSS, NAD+ seems to function as hydride acceptor in the first part of the reaction and subsequently as hydride donor in the second part. This was proved with chirally Cl-deuterated putrescines. Both a bacterial HSS (isolated from Rhodopseudomonas viridis) and the enzyme from PA-producing Eupatorium cannabinum transformed (R)-[1-2H]putrescine and (S)-[1-2H]-putrescine into homospermidine with a 100% retention of deuterium. Studies with deuterated and 14C-labeled substrates revealed that in the presence of putrescine spermidine is a substrate of HSS. The putrescine semialdehyde moiety of spermidine is combined with putrescine and 1,3-diaminopropane is released. Kinetic studies with plant HSS documented that the putrescine semialdehyde moiety of spermidine is incorporated into homospermidine with the same affinity (Km) and activity (Vmax) as putrescine. Plant HSS is highly specific for putrescine and spermidine; spermidine is not a substrate in the absence of putrescine. It is suggested that in the biosynthesis of homospermidine and thus PAs, one of the two C4 units is derived from spermidine and the other one from putrescine. A scheme of the HSS-catalyzed reaction is proposed that fully accounts for the enzymatic data and the results of previous tracer studies. Bacterial HSS is less substrate specific and not as well adapted as plant HSS to accept spermidine as a substrate.

Purification and characterization of homospermidine synthase in Acinetobacter tartarogenes ATCC 31105.

Homospermidine synthase, catalyzing the formation of homospermidine [H2N(CH2)4NH-(CH2)4NH2] from putrescine and NAD+ with concomitant liberation of NH3, was purified 600-fold over the crude extract with a yield of about 14% to homogeneity from Acinetobacter tartarogenes ATCC 31105. The enzyme had a native molecular mass of 102 kDa, with a pI of 5.0, and was apparently composed of two identical subunits (52 kDa), suggesting that a single protein catalyzes two serial reactions, oxidation of putrescine to 4-aminobutyraldehyde and subsequent reduction of the putative Schiff base formed between this aldehyde and a second molecule of putrescine to homospermidine. The Km values for putrescine and NAD+ were 280 and 18 microM, respectively. 1,3-Diaminopropane and cadaverine were inactive as substrates, and NAD+ could not be replaced by NADP+. 1,3-Diaminopropane and NADH were potent competitive inhibitors. The enzyme had a pH optimum of 8.7, was most active at 30 degrees C, and required K+ and dithiothreitol for full activity. Putrescine and NAD+ protected the enzyme from the inhibition by thiol reagents. The NH2-terminal amino acid sequence was AQWPVYGKISGPVVI. Some of these properties were compared with those of the homospermidine synthases from a photosynthetic bacterium, Rhodopseudomonas viridis and a plant, Lathyrus sativus.

Homospermidine synthase, the first pathway-specific enzyme in pyrrolizidine alkaloid biosynthesis

Root cultures of Senecio vulgaris accumulate high levels of radioactively labelled homospermidine (hspd) instead of pyrrolizidine alkaloids (PAs) when [ 14C]putrescine is fed together with β-hydroxyethylhydrazine (HEH), an inhibitor of diamine and polyamine oxidases. The endogenously accumulated hspd is exclusively incorporated into PAs when the HEH inhibition is released. There is no reversible link between hspd and the highly dynamic putrescine and spermidine pools. [ 14C]Pyrroline is readily taken up by the roots and is catabolized via 4-aminobutyric acid. Pyrroline is not incorporated into hspd or PAs. The results definitely exclude free pyrroline as an intermediate in PA biosynthesis and the participation of diamine oxidase or diamine transaminase in hspd formation. Homospermidine synthase (hspd synthase) is the first enzyme of the alkaloid specific pathway. Hspd synthase catalyses the formation of hspd from two moles putrescine in a NAD + dependent reaction. The enzyme from root cultures of Eupatorium cannabinum was partially purified and enriched 64-fold. The enzyme is labile, but could be stabilized by addition of spermidine and NAD +. The enzyme has a M r of 72 000, a pH optimum at 9, and the energy of activation is 54 kJ mol −1. The catalysed reaction is highly specific, NAD + cannot be substituted by NADP + and putrescine is the exclusive substrate. The K m values for putrescine and NAD + are 13.5 and 3.0 μM, respectively. The enzyme activity is competitively inhibited by 1,3-diaminopropane, spermidine and hspd with K i values of 6.3, 94 and 950 μM, respectively. Cadaverine ( K i 58 μM) and spermine ( K i 2.2 mM) are mixed-type inhibitors. The reaction is inhibited by NADH even at very low concentrations (2 μM). This indicates that the NADH formed in the first step of the overall reaction (i.e. oxidative deamination of the first putrescine moiety) remains enzyme-bound and serves as the electron donor of the second step (i.e. reduction of the assumed intermediate immine). Hspd and hspd synthase were detected in all root cultures (but not in dedifferentiated cell cultures), so far established from PA producing species of the family Asteraceae.

2516. Trichotoxin among the corn: Hou, C. T., Ciegler, A. & Hesseltine, C. W. (1972). New mycotoxin, trichotoxin A, from Trichoderma viride isolated from southern leaf blight-infected corn. Appl. Microbiol.23, 183

Three species of the Boraginaceae were studied: greenhouse-grown plants of Heliotropium indicum and Agrobacterium rhizogenes transformed roots cultures (hairy roots) of Cynoglossum officinale and Symphytum officinale. The species-specific pyrrolizidine alkaloid (PA) profiles of the three systems were established by GC–MS. All PAs are genuinely present as N-oxides. In H. indicum the tissue-specific PA distribution revealed the presence of PAs in all tissues with the highest levels in the inflorescences which in a flowering plant may account for more than 70% of total plant alkaloid. The sites of PA biosynthesis vary among species. In H. indicum PAs are synthesized in the shoot but not roots whereas they are only made in shoots for C. officinale and in roots of S. officinale. Classical tracer studies with radioactively labelled precursor amines (e.g., putrescine, spermidine and homospermidine) and various necine bases (trachelanthamidine, supinidine, retronecine, heliotridine) and potential ester alkaloid intermediates (e.g., trachelanthamine, supinine) were performed to evaluate the biosynthetic sequences. It was relevant to perform these comparative studies since the key enzyme of the core pathway, homospermidine synthase, evolved independently in the Boraginaceae and, for instance, in the Asteraceae [Reimann, A., Nurhayati, N., Backenkohler, A., Ober, D., 2004. Repeated evolution of the pyrrolizidine alkaloid-mediated defense system in separate angiosperm lineages. Plant Cell 16, 2772–2784.]. These studies showed that the core pathway for the formation of trachelanthamidine from putrescine and spermidine via homospermidine is common to the pathway in Senecio ssp. (Asteraceae). In both pathways homospermidine is further processed by a β-hydroxyethylhydrazine sensitive diamine oxidase. Further steps of PA biosynthesis starting with trachelanthamidine as common precursor occur in two successive stages. Firstly, the necine bases are structurally modified and either before or after this modification are converted into their O9-esters by esterification with one of the stereoisomers of 2,3-dihydroxy-2-isopropylbutyric acid, the unique necic acid of PAs of the lycopsamine type. Secondly, the necine O9-esters may be further diversified by O7- and/or O3′-acylation.