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 M_r 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.     

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.     

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.     

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.     

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.     

Formation of aminopropylhomospermidine from homospermidine in yeasts and thermophilic bacilli

Abstract When the yeasts Saccharomyces cerevisiae, Candida albicans and Schizosaccharomyces pombe and the thermophilic bacteria Bacillus stearothermophilus and Bacillus acidocaldarius were cultured in the presence of homospermidine, a new compound accumulated in the cells within a few days. This compound was identified as aminopropylhomospermidine [NH₂(CH₂)₃NH (CH₂)₄NH(CH₂)₄NH₂] by gas chromatographymass spectrometry (GC-MS) and by the enzymatic cleavage method developed in our laboratories. This polyamine was not produced from homospermidine in Escherichia coli, Bacillus subtilis, Bacillus alkalophilus , or a eukaryotic protozoon, Tetrahymena pyriformis , none of which usually contains appreciable amounts of spermine. These findings suggest that the synthesis of aminopropylhomospermidine from homospermidine is mediated by spermine synthase.     

Tissue distribution, core biosynthesis and diversification of pyrrolizidine alkaloids of the lycopsamine type in three Boraginaceae species

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 beta-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 O(9)-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 O(9)-esters may be further diversified by O(7)- and/or O(3')-acylation.     

Homospermidine in transgenic tobacco results in considerably reduced spermidine levels but is not converted to pyrrolizidine alkaloid precursors

Homospermidine synthase is the first specific enzyme in the biosynthesis of pyrrolizidine alkaloids. Whereas the substrates putrescine and spermidine are part of the highly dynamic polyamine pool of plants, the product homospermidine is incorporated exclusively into the necine base moiety of pyrrolizidine alkaloids. Recently, the gene encoding homospermidine synthase has been shown to have been recruited several times independently during angiosperm evolution by the duplication of the gene encoding deoxyhypusine synthase. To test whether high levels of homospermidine suffice for conversion, at least in traces, to precursors of pyrrolizidine alkaloids, transgenic tobacco plants were generated expressing homospermidine synthase. Analyses of the polyamine content revealed that, in the transgenic plants, about 80% of spermidine was replaced by homospermidine without any conspicuous modifications of the phenotype. Tracer-feeding experiments and gas chromatographic analyses suggested that these high levels of homospermidine were not sufficient to explain the formation of alkaloid precursors. These results are discussed with respect to current models of pathway evolution.     

Pyrrolizidine alkaloid biosynthesis,evolution of a pathway in plant secondary metabolism

The system of pyrrolizidine alkaloids has proven to be a powerful system for studying the evolution of a biosynthetic pathway in plant secondary metabolism. Pyrrolizidine alkaloids are typical plant secondary products produced by the plant as a defense against herbivores. The first specific enzyme, homospermidine synthase, has been shown to have evolved by duplication of the gene encoding deoxyhypusine synthase, which is involved in primary metabolism. Despite the identical function of homospermidine synthase for pyrrolizidine alkaloid biosynthesis in the various plant lineages, this gene duplication has occurred several times independently during angiosperm evolution. After duplication, these gene copies diverged with respect to gene function and regulation. In the diverse plant lineages producing pyrrolizidine alkaloids, homospermidine synthase has been shown to be expressed in a variety of tissues, suggesting that the regulatory elements were recruited individually after the duplication of the structural gene. The molecular, kinetic, and expression data of this system are discussed with respect to current models of gene and pathway evolution.     

Homospermidine synthase contributes to salt tolerance in free-living Rhizobium tropici and in symbiosis with Phaseolus vulgaris

Plant and Soil - Homospermidine is known to be the most abundant polyamine in root nodules of Phaseolus vulgaris induced by Rhizobium tropici. In addition, homospermidine is involved in the stress...     

Population decline despite high genetic diversity in the new allopolyploid species Senecio cambrensis (Asteraceae)

Senecio cambrensis (Welsh groundsel) is a new allohexaploid species, which originated in Wales, UK, in the early part of the 20th century following hybridization between the native tetraploid groundsel (Senecio vulgaris) and the introduced diploid Oxford ragwort (Senecio squalidus). A survey of the number of populations and flowering individuals per population of S. cambrensis in Wales was conducted at peak flowering time in June 2002, 2003 and 2004. The results show a dramatic decrease in both population number and population size of the species since the 1980s when the last population census was conducted. A survey of amplified fragment length polymorphism (AFLP) variation showed that this decline has occurred despite the fact that S. cambrensis contains a high level of genetic diversity with each individual screened possessing a unique multilocus phenotype. The level of variance within the species was similar to that found in one parent (S. vulgaris) and slightly greater than that among samples of the other parent (S. squalidus). Only a small proportion (5%) of AFLP diversity was partitioned among populations indicating a lack of population structure and possibly high levels of gene flow via seed dispersal in what is predominantly a selfing species. Senecio cambrensis showed closer similarity in AFLP phenotype to S. vulgaris than to S. squalidus. Possible causes of this and also the high level of AFLP diversity found in S. cambrensis are discussed. It is suggested that intergenomic recombination following occasional multivalent formation during meiosis in S. cambrensis is likely to be an important cause of both phenomena, although other causes are not ruled out.     

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.     

Elucidative studies on the generic concept of Senecio (Asteraceae)

VINCENT, P. L. D. & GETLIFFE, F. M., 1992. Elucidative studies on the generic concept of Senecio (Asteraceae) . This paper presents the results of studies of the generic concept of Senecio sensu stricto. The sample of taxa studied consisted of 93 Natal senecios (including seven varieties and two forms), five Cape heterochromous senecios and nine non-southern African senecios, including the type of the genus, S. vulgaris L. Also included in the study were six species from taxonomically closely related genera in the tribe Senecioneae and one Senecio of uncertain taxonomic position. The phenotype of these taxa was investigated with respect to a large number (122) of morphological and micromorphological characters. Six characters were selected as being taxonomically important with respect to elucidating the generic concept of Senecio sensu stricto. The generic concept of Senecio has been provisionally re-circumscribed and the generic status of each of the senecios and non-senecios studied has been tested according to this concept of Senecio sensu stricto sensu Vincent. In the light of this concept of Senecio , the following species are recommended for exclusion from Senecio sensu stricto sensu Vincent: S. cissampelinus, S. transvaalensis, S. syringifolius and S. hockii. The following species are considered to be peripheral to Senecio sensu stricto sensu Vincent: S. tanacetopsis, S. seminiveus, S. medley-woodii, S. tamoides, S. helminthioides, S. barbertonicus, S. brevilorus, S. viminalis, S. radicans and S.fulgens. Before any taxonomic changes are made to the current composition of Senecio , the concept of Senecio sensu stricto sensu Vincent, is being tested on a worldwide sample of the genus.     

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.     

The evolution of pyrrolizidine alkaloid biosynthesis and diversity in the Senecioneae

Pyrrolizidine alkaloids are characteristic secondary metabolites of the Asteraceae and some other plant families. They are especially numerous and diverse in the tribe Senecioneae and form a powerful defense mechanism against herbivores. Studies into the evolution of pyrrolizidine alkaloid biosynthesis using Senecio species have identified homospermidine synthase as the enzyme responsible for the synthesis of the first specific intermediate. These studies further indicated that the homospermidine synthase-encoding gene was recruited following gene duplication of deoxyhypusine synthase and that this occurred independently in several different angiosperm lineages. A review of published pyrrolizidine alkaloid data shows that the Senecioneae are characterized by a large qualitative and quantitative variation in pyrrolizidine alkaloid profiles and that these data demonstrate little phylogenetic signal. This suggests that although the first steps of this pathway are highly conserved, the diversification of secondarily derived pyrrolizidine alkaloids is extremely plastic.     

Evidence for the presence of an F-type ATP synthase involved in sulfate respiration in Desulfovibrio vulgaris

Using a library of genomic DNA from Desulfovibrio vulgaris Miyazaki F, a strict anaerobe, and two synthetic deoxyoligonucleotide probes designed for F-type ATPases, the genes for open reading frames (ORFs) 1 to 5 were cloned and sequenced. The predicted protein sequences of the gene products indicate that they are composed of 172, 488, 294, 471, and 134 amino acids, respectively, and that they share considerable identity at the amino acid level with delta, alpha, gamma, beta, and epsilon subunits found in other F-type ATPases, respectively. Furthermore, a component carrying ATPase activity was partially purified from the cytoplasmic membrane fraction of the D. vulgaris Miyazaki F cells. The N-terminal amino acid sequences of three major polypeptides separated by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis were identical to those of the products predicted by the sequences of ORF-2, ORF-3, and ORF-4, suggesting that an F-type ATPase is functioning in the D. vulgaris Miyazaki F cytoplasmic membrane. The amount of the F-type ATPase produced in the D. vulgaris Miyazaki F cells is similar to that in the Escherichia coli cells cultured aerobically. It indicates that the enzyme works as an ATP synthase in the D. vulgaris Miyazaki F cells in connection with sulfate respiration.     

Introduction of the Escherichia coli gdhA gene into Rhizobium phaseoli: effect on nitrogen fixation.

Rhizobium phaseoli lacks glutamate dehydrogenase (GDH) and assimilates ammonium by the glutamine synthetase-glutamate synthase pathway. A strain of R. phaseoli harboring the Escherichia coli GDH structural gene (gdhA) was constructed. GDH activity was expressed in R. phaseoli in the free-living state and in symbiosis. Nodules with bacteroids that expressed GDH activity had severe impairment of nitrogen fixation. Also, R. phaseoli cells that lost GDH activity and assimilated ammonium by the glutamine synthetase-glutamate synthase pathway preferentially nodulated Phaseolus vulgaris.