Spermine is not essential for growth of Saccharomyces cerevisiae: identification of the SPE4 gene (spermine synthase) and characterization of a spe4 deletion mutant

Spermine, ubiquitously present in most organisms, is the final product of the biosynthetic pathway for polyamines and is synthesized from spermidine. In order to investigate the physiological roles of spermine, we identified the SPE4 gene, which codes for spermine synthase, on the right arm of chromosome XII of Saccharomyces cerevisiae and prepared a deletion mutant in this gene. This mutant has neither spermine nor spermine synthase activity. Using the spe4 deletion mutant, we show that S. cerevisiae does not require spermine for growth, even though spermine is normally present in the wild-type organism. This is in striking contrast to the absolute requirement of S. cerevisiae for spermidine for growth, which we had previously reported using a mutant lacking the SPE3 gene (spermidine synthase) [Hamasaki-Katagiri, N., Tabor, C.W., Tabor, H., 1997. Spermidine biosynthesis in Saccharomyces cerevisiae: Polyamine requirement of a null mutant of the SPE3 gene (spermidine synthase). Gene 187, 35–43].     

Polyamine oxidase of millet shoots

Polyamine oxidase was purified ca 168-fold from the acetone powder extract of millet shoots. The light yellow enzyme had maximum absorption at 278,380 and 460 nm. The absorption at 380 and 460 nm was decreased by the addition of spermidine. The enzyme (M, ca 80 000) showed a high specificity for spermine and spermidine (K_ms 6 × 10⁻⁵ M and 5 × 10⁻⁷ M respectively). The enzyme was inhibited by quinacrine and acriflavine.     

Polyamines are necessary for maximum in vitro synthesis of globin peptides and play a role in chain initiation.

The salt wash fraction removed from rabbit reticulocyte ribosomes with 0.5 m KCl contains dialyzable components required for maximum in vitro synthesis of globin peptides. The active substances were identified as spermidine and spermine. Rabbit reticulocyte ribosomes contain spermine and spermidine in a 1:3 ratio of which about 75% is removed in the 0.5 m KCl wash fraction. Dialyzed salt wash can be reactivated for in vitro protein synthesis by addition of either spermine, spermidine, or Mg²⁺ ion. A twofold higher leucine incorporation into protein was obtained with the optimum concentration of either polyamine than with Mg²⁺. Spermidine is effective in lowering the Mg²⁺ requirement for initiation of phenylalanine peptides in the poly(U)-directed system, apparently by formation of an initiation complex. Also, spermidine competitively interferes with edeine inhibition of globin chain initiation. These results indicate that spermidine may play a special role in peptide initiation.     

Polyamine metabolism is involved in adipogenesis of 3T3-L1 cells

Polyamines spermidine and spermine are known to be required for mammalian cell proliferation and for embryonic development. Alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC) a limiting enzyme of polyamine biosynthesis, depleted the cellular polyamines and prevented triglyceride accumulation and differentiation in 3T3-L1 cells. In this study, to explore the function of polyamines in adipogenesis, we examined the effect of polyamine biosynthesis inhibitors on adipocyte differentiation and lipid accumulation of 3T3-L1 cells. The spermidine synthase inhibitor trans-4-methylcyclohexylamine (MCHA) increased spermine/spermidine ratios, whereas the spermine synthase inhibitor N-(3-aminopropyl)-cyclohexylamine (APCHA) decreased the ratios in the cells. MCHA was found to decrease lipid accumulation and GPDH activity during differentiation, while APCHA increased lipid accumulation and GPDH activity indicating the enhancement of differentiation. The polyamine-acetylating enzyme, spermidine/spermine N ¹-acetyltransferase (SSAT) activity was increased within a few hours after stimulus for differentiation, and was found to be elevated by APCHA. In mature adipocytes APCHA decreased lipid accumulation while MCHA had the opposite effect. An acetylpolyamine oxidase and spermine oxidase inhibitor MDL72527 or an antioxidant N-acetylcysteine prevented the promoting effect of APCHA on adipogenesis. These results suggest that not only spermine/spermidine ratios but also polyamine catabolic enzyme activity may contribute to adipogenesis.     

Dicyclohexylamine-induced shift of biosynthesis from spermidine to spermine in plant protoplasts

An improved analytical method, based on high pressure liquid chromatography, has been developed for the simultaneous determination of the polyamines and S-adenosyl-containing compounds in extracts of plant protoplasts. The method involves simple procedures for sample preparation and permits quantification of 1 picomole or less for all the compounds. This method has been used to study the effects of dicyclohexylamine, an inhibitor of plant spermidine synthase (Sindhu, R. K., S. S. Cohen 1984 Plant Physiol 74: 645-649), on biosynthesis of polyamines and 1-aminocyclopropane-1-carboxylate in protoplasts derived from Chinese cabbage leaves. Dicyclohexylamine effectively inhibits spermidine synthase in vivo. Inhibition of the synthesis of spermidine by dicyclohexylamine resulted in a stimulation of spermine synthesis, without significant effect on the synthesis of 1-aminocyclopropane-1-carboxylate. Decarboxylated S-adenosylmethionine is present in control Chinese cabbage protoplasts at ~10⁻¹⁸ moles per cell, and dicyclohexylamine caused an increase of this metabolite of up to 10-fold in a 4-hour period. The increase in decarboxylated S-adenosylmethionine permitted an increased synthesis of spermine. These findings suggest that the availability of decarboxylated S-adenosylmethionine may be rate-limiting for the synthesis of spermine in plant protoplasts.     

Novel metabolically stable and functionally active mimetic of spermidine

Earlier unknown 1,8-diamino-3-methyl-4-azanonane (γ-MeSpd) was synthesized. The analogue was a substrate of neither spermine/spermidine N ¹-acetyltransferase nor spermine synthase, but was capable to support the growth of DU145 cells having depleted polyamine pools. Such a combination of γ-MeSpd properties discloses novel opportunities to study cellular functions of catabolically unstable and easily interconvertible spermine and spermidine.     

Further properties of the polyamine oxidase from oat seedlings

After purification, the polyamine oxidase from the leaves of oat seedlings grown in the dark appeared to be homogeneous on electrophoresis. The MW determined by density gradient centrifugation was 119 000. The enzyme would not oxidise diaminodipropylamine and neither diaminodipropylamine nor diaminopropane were inhibitors at concentrations up to 1 mM. With spermidine as substrate, the energy of activation was 19.7 kJ/mol and activity was reduced to 50% on heating for 10 min at 50°. With spermine as substrate, activity was increased up to 3-fold in the presence of M sodium chloride. This stimulation was not observed with spermidine as substrate The enzyme was also stimulated by sodium phosphate and sodium citrate at high concentrations. The pH for optimal stability was 6.5, the same as the pH for maximum activity with both spermidine and spermine as substrates. For spermidine and spermine the K_ms were 8 × 10 ^?6 M and 2 × 10 ^?6 M respectively. Loss of activity on storage of leaves at ? 15° was ca 5 % per week and in extracts the loss was ca 10 % per week.     

Purification and properties of agmatine iminohydrolase from groundnut cotyledons

Agmatine iminohydrolase was purified ca 375-fold from groundnut cotyledons. The enzyme exhibited an optimum pH between 5.5 and 8.5 and the energy of activation was 22 kcal/mol. The K_m for agmatine was (7.57 ± 0.77) × 10^?4 M. The enzyme was inhibited by tryptamine, putrescine, cadaverine, spermidine and spermine. Inhibition by cadaverine and spermidine was competitive. The K_i values for cadaverine and spermidine were 4.1 × 10^?3 and 7.5 × 10^?4 M, respectively.     

Identification and characterization of a novel spermidine/spermine acetyltransferase encoded by gene ste26 from Streptomyces sp. 139

Streptomyces sp. 139 produces a novel exopolysaccharide (EPS) designated Ebosin which can bind IL-1R specifically and exhibits anti-rheumatic arthritis activity in vivo. With the Ebosin biosynthesis gene cluster (ste) consisting of 27 ORFs identified previously the focus of this study was to characterize the protein encoded by ste26 gene. After cloning and expressing ste26 in Escherichia coli BL21, we purified the recombinant Ste26 protein and revealed its ability of transferring the acetyl group from AcCoA to spermidine and spermine, with spermine being the preferred substrate. Therefore Ste26 has been determined to be a spermidine/spermine acetyltransferase which can use spermine (Km of 72.1 ± 7.4 μM), spermidine (Km of 147.2 ± 11 μM), AcCoA (Km of 45.7 ± 2.5 μM) and poly-l-lysine (Km of 99.7 ± 11 μM) as substrates. The optimum pH, temperature and time for the activity have been shown to be 7.5, 37°C and 10 min, respectively. This is the first spermidine/spermine acetyltransferase characterized in Streptomyces and its function in Ebosin biosynthesis is discussed.     

Inhibition of the synthesis of polyamines and macromolecules by 5′-methylthioadenosine and 5′-alkylthiotubercidins in BHK21 cells

5′-Methylthioadenosine and four 5′-alkylthiotubercidins were tested for their ability to inhibit polyamine synthesis in vitro and to decrease polyamine concentration and prevent growth of baby-hamster-kidney (BHK21) cells. 5′-Methylthioadenosine and 5′-methylthiotubercidin decreased the activity of spermidine synthase from brain to roughly the same extent, whereas brain spermine synthase was much more strongly inhibited by 5′-methylthioadenosine compared with 5′-methylthiotubercidin. These nucleoside derivatives also inhibited the growth of BHK21 cells and increased the concentration of putrescine. 5′-Methylthioadenosine decreased cellular spermine concentration, whereas 5′-methylthiotubercidin lowered the concentration of spermidine. The activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase were enhanced in cells grown in the presence of 5′-methylthiotubercidin. The growth inhibition produced by these nucleoside derivatives was not reversed by exogenous spermidine or spermine. 5′-Ethylthiotubercidin, 5′-propylthiotubercidin and 5′-isopropylthiotubercidin did not appreciably inhibit spermidine or spermine synthase in vitro or decrease the cellular polyamine content, but effectively prevented the growth of BHK21 cells. All nucleoside derivatives at concentrations of 0.2–1 mm caused a rapid inhibition of protein synthesis. It is concluded that the growth inhibition produced by 5′-methylthioadenosine and 5′-alkylthiotubercidins was not primarily due to polyamine depletion but other target sites, for instance the cellular nucleotide pool, cell membranes etc. must be considered.     

Influence of plant growth regulators, polyamines and glycerol interaction on growth and morphogenesis of carposporelings of Grateloupia cultured in vitro

The influence of the plant growth regulators 2,4-D, GA₃, BA and kinetin, and the polyamines putrescine, spermidine and spermine were tested on axenic in vitro cultures of carposporelings of Grateloupia doryphora. The auxin 2,4-D (10^-3 M) and the polyamine spermine (10^-6 M and 10^-3 M) induced a callus (disorganised cell mass that arose from the organised tissue of the carposporeling, as demonstrated by microscopic monitoring of the tissue). Putrescine and spermidine (10^-3 M) transformed the carposporelings into cell masses that produced shoots. BA (10^-3 M) and kinetin (10^-6 M and 10^-3 M) were inhibitory. In 10^-1 M glycerol-containing culture medium, which is known to induce the formation of morphogenic cell masses, the addition of GA₃ M) resulted in the inhibition of the morphogenesis (i.e. shoot emission) in the cell mass. The kinetin at 10^-6 M inhibited morphogenesis, whilst at 10^-3 M inhibited even the formation of the cell masses. The combination of glycerol (10^-1 M) and the auxin 2,4-D (10^-6 and 10^-3 M) or the polyamines putrescine, spermidine and spermine (10^-6 and 10^-3 M) resulted in a bigger size of the cell masses that led to a higher amount of shoots per cell mass than in glycerol alone. This revised version was published online in June 2006 with corrections to the Cover Date.     

Polyamine catabolism in carcinogenesis: potential targets for chemotherapy and chemoprevention

Polyamines, including spermine, spermidine, and the precursor diamine, putrescine, are naturally occurring polycationic alkylamines that are required for eukaryotic cell growth, differentiation, and survival. This absolute requirement for polyamines and the need to maintain intracellular levels within specific ranges require a highly regulated metabolic pathway primed for rapid changes in response to cellular growth signals, environmental changes, and stress. Although the polyamine metabolic pathway is strictly regulated in normal cells, dysregulation of polyamine metabolism is a frequent event in cancer. Recent studies suggest that the polyamine catabolic pathway may be involved in the etiology of some epithelial cancers. The catabolism of spermine to spermidine utilizes either the one-step enzymatic reaction of spermine oxidase (SMO) or the two-step process of spermidine/spermine N ¹-acetyltransferase (SSAT) coupled with the peroxisomal enzyme N ¹-acetylpolyamine oxidase. Both catabolic pathways produce hydrogen peroxide and a reactive aldehyde that are capable of damaging DNA and other critical cellular components. The catabolic pathway also depletes the intracellular concentrations of spermidine and spermine, which are free radical scavengers. Consequently, the polyamine catabolic pathway in general and specifically SMO and SSAT provide exciting new targets for chemoprevention and/or chemotherapy.     

Adenosylmethionine decarboxylase from various organisms: Relation of the putrescine activation of the enzyme to the ability of the organism to synthesize spermine

S-adenosyl-L-methionine decarboxylase (EC 4.1.1.50) from most eukaryotic organisms is activated by putrescine whereas the corresponding enzyme from bacterial sources shows a stringent requirement for magnesium ions. Adenosylmethionine decarboxylase from lower eukaryotes such as protozoa, however, is not influenced by diamines, neither are any metals needed for its maximal activity. A common characteristic of those organisms containing putrescine-insensitive adenosylmethionine decarboxylase appeared to be either a total absence or very low intracellular content of spermine. While extracts of all organisms containing putrescine-activated adenosylmethionine decarboxylase (animal tissues and yeast) exhibited easily measurable spermine synthase activity, no such activity was detected in cells of Tetrahymena pyriformis, Escherichia coli or Pseudomonas aeruginosa all containing adenosylmethionine decarboxylase insensitive to putrescine and other diamines.The activation of adenosylmethionine decarboxylase by putrescine, the immediate precursor of spermidine, may thus assure the availability of sufficient amounts of decarboxylated adenosylmethionine (S-methyladenosyl-cysteamine) for the synthesis of spermidine even in the presence of a spermine synthesizing system competing for the same precursor (decarboxylated adenosylmethionine).     

Polyamines in testosterone-induced hypertrophic and antifolate-induced hyperplastic mouse kidney. Differential effect of α-difluoromethylornithine

In the testosterone-induced hypertrophic and antifolate (N¹⁰-propargyl,5,6-dideazafolic acid, CB 3717)-induced hyperplastic mouse kidney models, a marked increase of two diamine levels — putrescine and cadaverine — occurred which paralled induced ornithine decarboxylase (ODC) activity. Under these conditions the augmentation of spermidine levels was much smaller, while spermine levels were affected differentially — increased by testosterone and decreased by CB 3717; this resulted in an increase of spermidine/spermine ratio in hyperplastic, but not hypertrophic kidney. α-Difluoromethylornithine (DFMO) prevented testosterone- or CB 3717-induced increment of both diamine levels. Spermidine and spermine depletion in response to DFMO was significant in hyperplastic kidney only. DFMO also significantly affected the other biochemical markers of hyperplasia, namely lowered CB 3717-induced cell proliferation rate and increased S-adenosylmethionie decarboxylase (AdoMetDC) activity. In contrast, testosterone-induced hypertrophy was not influenced by DFMO, as judged by the lack of its effect on S-adenosylmethionine synthetase and cystathionine synthase activity. These results indicate that the increase of putrescine levels does not mediate testosterone-induced renal hypertrophy and possibly also antifolate-induced hyperplasia. The involvement of spermidine in mediation of renal hyperplasia is highly possible, while that of spermine is excluded.     

Consecutive action of two BAHD acyltransferases promotes tetracoumaroyl spermine accumulation in chicory

Fully substituted phenolamide accumulation in the pollen coat of Eudicotyledons is a conserved evolutionary chemical trait. Interestingly, spermidine derivatives are replaced by spermine derivatives as the main phenolamide accumulated in the Asteraceae family. Here, we show that the full substitution of spermine in chicory (Cichorium intybus) requires the successive action of two enzymes, that is spermidine hydroxycinnamoyl transferase-like proteins 1 and 2 (CiSHT1 and CiSHT2), two members of the BAHD enzyme family. Deletion of these genes in chicory using CRISPR/Cas9 gene editing technology evidenced that CiSHT2 catalyzes the first N-acylation steps, whereas CiSHT1 fulfills the substitution to give rise to tetracoumaroyl spermine. Additional experiments using Nicotiana benthamiana confirmed these findings. Expression of CiSHT2 alone promoted partially substituted spermine accumulation, and coexpression of CiSHT2 and CiSHT1 promoted synthesis and accumulation of the fully substituted spermine. Structural characterization of the main product of CiSHT2 using nuclear magnetic resonance revealed that CiSHT2 preferentially catalyzed N-acylation of secondary amines to form N⁵,N¹⁰-dicoumaroyl spermine, whereas CiSHT1 used this substrate to synthesize tetracoumaroyl spermine. We showed that spermine availability may be a key determinant toward preferential accumulation of spermine derivatives over spermidine derivatives in chicory. Our results reveal a subfunctionalization among the spermidine hydroxycinnamoyl transferase that was accompanied by a modification of free polyamine metabolism that has resulted in the accumulation of this new phenolamide in chicory and most probably in all Asteraceae. Finally, genetically engineered yeast (Saccharomyces cerevisiae) was shown to be a promising host platform to produce these compounds.

Emergence of an unusual chemical signature in the chicory pollen coat relies on subfunctionalization of CiSHT1 and CiSHT2.     

Characterization of transgenic mice with overexpression of spermidine synthase

A composite cytomegalovirus-immediate early gene enhancer/chicken β-actin promoter (CAG) was utilized to generate transgenic mice that overexpress human spermidine synthase (SpdS) to determine the impact of elevated spermidine synthase activity on murine development and physiology. CAG-SpdS mice were viable and fertile and tissue SpdS activity was increased up to ninefold. This increased SpdS activity did not result in a dramatic elevation of spermidine or spermine levels but did lead to a 1.5- to 2-fold reduction in tissue spermine:spermidine ratio in heart, muscle and liver tissues with the highest levels of SpdS activity. This new mouse model enabled simultaneous overexpression of SpdS and other polyamine biosynthetic enzymes by combining transgenic animals. The combined overexpression of both SpdS and spermine synthase (SpmS) in CAG-SpdS/CAG-SpmS bitransgenic mice did not impair viability or lead to overt developmental abnormalities but instead normalized the elevated tissue spermine:spermidine ratios of CAG-SpmS mice. The CAG-SpdS mice were bred to MHC-AdoMetDC mice with a >100-fold increase in cardiac S-adenosylmethionine decarboxylase (AdoMetDC) activity to determine if elevated dcAdoMet would facilitate greater spermidine accumulation in mice with SpdS overexpression. CAG-SpdS/MHC-AdoMetDC bitransgenic animals were produced at the expected frequency and exhibited cardiac polyamine levels comparable to MHC-AdoMetDC littermates. Taken together these results indicate that SpdS levels are not rate limiting in vivo for polyamine biosynthesis and are unlikely to exert significant regulatory effects on cellular polyamine content and function.     

Kinetic studies on inhibition of aminopropyltransferases by aurintricarboxylic acid in vitro

Activities of aminopropyltransferases (spermidine synthase and spermine synthase) were inhibited by aurintricarboxylic acid (ATA). Spermidine synthase was slightly more sensitive to the inhibitor than spermine synthase. These inhibitions were not prevented by 0.15 M NaCl. Inhibition by ATA of spermidine synthase was ‘uncompetitive’ with respect to putrescine and that of spermine synthase was ‘non-competitive’ with respect to spermidine. When the amount of spermidine synthase or spermine synthase was varied, inhibition ratio hardly changed on either case implying no appreciable interaction between ATA and these enzymes.