Spermidine biosynthesis in Saccharomyces cerevisiae: Polyamine requirement of a null mutant of the SPE3 gene (spermidine synthase)

The Saccharomyces cerevisiae SPE3 gene, coding for spermidine synthase, was cloned, sequenced, and localized on the right arm of chromosome XVI. The deduced amino acid sequence has a high similarity to mammalian spermidine synthases, and has putative S-adenosylmethionine binding motifs. To investigate the effect of total loss of the SPE3 gene, we constructed a null mutant of this gene, spe3Δ, which has no spermidine synthase activity and has an absolute requirement for spermidine or spermine for the growth. This requirement is satisfied by a very low concentration of spermidine (10⁻⁸ M) or a higher concentration of spermine (10⁻⁶ M).     

黄瓜花性别分化与内源多胺的关系

研究了黄瓜雌、雄花几个主要发育时期和性别逆转过程中内源多胺的变化。结果表明 ,雄花在不同发育时期 ,内源腐胺含量均高于雌花 ,腐胺含量的显著升高伴随着花粉粒的形成 ,高腐胺含量是雄花发育的特征。雌花在大孢子母细胞时期以后直到雌花发育成熟 ,其内源尸胺含量均高于雄花 ,高尸胺含量可能有利于雌花的发育。高水平的内源多胺、精胺和亚精胺可能有利于雌花大孢子母细胞的形成。亚精胺和腐胺含量随着大小孢子四分体形成和大孢子核的连续分裂而分别表现下降和上升。雌性系黄瓜经硝酸银诱导雄花处理后 ,茎尖内源亚精胺含量下降 ,腐胺含量上升 ,从而诱导雄花形成 ;雄性系黄瓜经乙烯利诱导雌花处理后 ,茎尖内源亚精胺含量上升 ,腐胺含量下降 ,从而诱导雌花形成     

Putrescine N-methyltransferase – The start for alkaloids

Putrescine N-methyltransferase (PMT) catalyses S-adenosylmethionine (SAM) dependent methylation of the diamine putrescine. The product N-methylputrescine is the first specific metabolite on the route to nicotine, tropane, and nortropane alkaloids. PMT cDNA sequences were cloned from tobacco species and other Solanaceae, also from nortropane-forming Convolvulaceae and enzyme proteins were synthesised in Escherichia coli. PMT activity was measured by HPLC separation of polyamine derivatives and by an enzyme-coupled colorimetric assay using S-adenosylhomocysteine. PMT cDNA sequences resemble those of plant spermidine synthases (putrescine aminopropyltransferases) and display little similarity to other plant methyltransferases. PMT is likely to have evolved from the ubiquitous enzyme spermidine synthase. PMT and spermidine synthase proteins share the same overall protein structure; they bind the same substrate putrescine and similar co-substrates, SAM and decarboxylated S-adenosylmethionine. The active sites of both proteins, however, were shaped differentially in the course of evolution. Phylogenetic analysis of both enzyme groups from plants revealed a deep bifurcation and confirmed an early descent of PMT from spermidine synthase in the course of angiosperm development.     

Effect of low dose of spermidine on physiological changes in salt-stressed cucumber plants

The present study was aimed to assess the ameliorative potentiality of exogenously applied low dose of spermidine (Spd) (4.0 mL of 0.1 mM) against salt stress in cucumber (Cucumis sativus L.) plants. Salt stress inhibited plant growth, while Spd increased the shoot length and dry weight of leaves in salt-stressed plants. Chlorophyll, carotenoids, and sucrose contents were lower, and the accumulation of superoxide radical was higher in salt-affected plants than in controls, and these detrimental effects were mitigated by Spd treatment. Moreover, salinity diminished the reduced glutathione and total polyphenols and inhibited the activities of catalase, peroxidase, and polyphenol oxidase as compared with controls, and Spd treatment increased all antioxidant activities in salt-injured plants. NaCl-induced oxidative stress caused a significant decrease in GA₄ and GA₅ contents. Spd treatment ameliorated these salt stress effects by increasing the quantities of GA₄. In addition, sodium content was higher and calcium content was lower in salt-treated plants, while Spd treatment reduced the sodium accumulation and increased the calcium level in plants exposed to NaCl. The results suggest that exogenous application of low Spd dose can ameliorate the salt stress effects on cucumber by modulating the components of photosynthetic pigments, antioxidants, gibberellins, and minerals.     

Crystal structures and enzymatic properties of a triamine/agmatine aminopropyltransferase from Thermus thermophilus

To maintain functional conformations of DNA and RNA in high-temperature environments, an extremely thermophilic bacterium, Thermus thermophilus, employs a unique polyamine biosynthetic pathway and produces more than 16 types of polyamines. In the thermophile genome, only one spermidine synthase homolog (SpeE) was found and it was shown to be a key enzyme in the pathway. The catalytic assay of the purified enzyme revealed that it utilizes triamines (norspermidine and spermidine) and agmatine as acceptors in its aminopropyl transfer reaction; therefore, the enzyme was denoted as a triamine/agmatine aminopropyltransferase (TAAPT). We determined the crystal structures of the enzyme complexed with and without the aminopropyl group donor S-adenosylmethionine. Despite sequence and structural similarity with spermidine synthases from other organisms, a novel C-terminal β-sheet and differences in the catalytic site were observed. The C-terminal module interacts with the gatekeeping loop and fixes the open conformation of the loop to recognize larger polyamine substrates such as agmatine and spermidine. Additional computational docking studies suggest that the structural differences of the catalytic site also contribute to recognition of the aminopropyl/aminobutyl or guanidium moiety of the substrates of TAAPT. These results explain in part the extraordinarily diverse polyamine spectrum found in T. thermophilus.     

Sucrose metabolism and accumulation in developing fruit of Cucumis

Sucrose, reducing sugars and starch content were measured in developing cucumber (C. sativus cv Delilah), sweet melon (C. melon cv Galia and cv Noy Yizre'el) and non-sweet melon (C. melo cv Bird's Nest) fruit. Sweet melon were characterized by accumulation of sucrose in the maturing fruit, while cucumber and non-sweet melon had a low sucrose content at all stages studied. Soluble acid invertase activity (EC 3.2.1.26) dramatically decreased in sweet melon, concomitant with the onset of sucrose accumulation. Significant activity of soluble acid invertase was retained in mature cucumber and non-sweet melon. Insoluble acid invertase, determined not to be an artifact of extraction, constituted a significant portion of total invertase activity (ca 25% in young sweet melon and ca 50% in young cucumber). In sweet melon sucrose synthase activity (EC 2.4.1.13), measured in both the cleavage and synthesis direction, increased during the sucrose accumulation period. The results are discussed in terms of the roles of invertase and sucrose synthase in sucrose accumulation in Cucumis.     

Piriformospora indica promotes cucumber tolerance against Root-knot nematode by modulating photosynthesis and innate responsive genes

Root Knot Nematode (RKN, Meloidogyne incognita) is one of the greatest damaging soil pathogens causes severe yield losses in cucumber and many other economic crops. Here, we evaluated the potential antagonistic effect of the root mutualistic fungus Piriformospora indica against RKN and their impact on vegetative growth, yield, photosynthesis, endogenous salicylic acid (SA) and its responsive genes. Our results showed that P. indica dramatically decreased the damage on shoot and root architecture of cucumber plants, which consequently enhanced yield of infested plants. Likewise, P. indica colonization clearly improved the chlorophyll content and delimited the negative impact of RNK on photosynthesis. Moreover, P. indica colonization exhibited a significant reduction of different vital nematological parameters such as soil larva density, amount of eggs/eggmass, eggmasses, females and amount of galls at cucumber roots. Additionally, the results showed that SA level was significantly increased generally in the roots of all treatments especially in plants infested with RKN alone as compared to control. This suggests that P. indica promoting SA levels in host cucumber plant roots to antagonize the RKN and alleviate severity damages occurred in its roots. This higher levels of SA in cucumber roots was consistent with the higher expressional levels of SA pathway genes PR1 and PR3. Furthermore, P. indica colonization reduces PR1, PR3 and increased NPR1 in roots of RKN infested cucumber plants when compared to non-colonized plants. Interestingly, our in vitro results showed that direct application of P. indica suspension against the J2s exhibited a significant increase in mortality ratio. Our results collectively suggest that P. indica promoting morphological, physiological and SA levels that might together play a major important role to alleviate the adverse impact of RKN in cucumber.     

外源亚精胺对淹涝胁迫下黄瓜根HSP70表达的影响

采用逆转录聚合酶链式反应(RT-PCR)及蛋白免疫印迹杂交(Western Blot)技术,研究0.5 mmol/L亚精胺浸种的黄瓜幼苗在淹水胁迫下,根热激蛋白70基因(HSP70)mRNA和蛋白质的表达量的变化。结果表明:淹水胁迫使黄瓜根HSP70的mRNA和蛋白的表达呈现先上升后下降的趋势,在淹水4 h时,HSP70的mRNA和蛋白表达量均极显著高于未淹水处理; 亚精胺浸种的黄瓜根HSP70的mRNA和蛋白的表达量在24 h内呈一直上升的趋势,在淹水24 h时,HSP70的mRNA和蛋白表达量均极显著高于未淹水处理。淹涝胁迫下,亚精胺浸种的黄瓜根HSP70的mRNA和蛋白表达量在淹水12 h和24 h时极显著高于蒸馏水浸种。外源亚精胺能诱导淹涝胁迫下黄瓜幼苗根HSP70 mRNA和蛋白质的表达量的增加,缓解淹涝胁迫对黄瓜造成的伤害。     

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.     

Role of polyamines in the cytokinin-dependent physiological processes II. Modulation of polyamine levels during cytokinin-stimulated expansion of cucumber cotyledons

The cucumber cotyledon expansion test was used as a model system to study a possible relationship between cytokinin and polyamines. When kinetin was applied to excised cotyledons incubated in the dark it caused a marked increase in the activity of arginine decarboxylase. As a result of ADC action, putrescine content also rose markedly, whereas the level of spermidine and spermine decreased. However, inhibition of putrescine biosynthesis with D-arginine did not affect cytokinin promotion growth. Applied alone, putrescine had no significant effect on growth. These results indicate that the large increase in putrescine content that derives from cytokinin treatment cotyledons is not essential for cytokinin-induced expansion of cotyledons. Addition of K⁺ and Ca²⁺ ions to the cotyledons incubated with cytokinin caused a marked reduction in the putrescine level and ADC activity. The higher level of putrescine (35 %) and spermine (62 %) bound to chromatin and the large increase (174 %) in spermidine content bound to ribosomes which derive from cytokinintreated cotyledons in relation to literature data can indicate that these polyamines may play an important role in gene expression during cytokinin-stimulated expansion of cucumber cotyledons. The inhibition of cytokinin effect, viz. enlargement of the cotyledons by inhibitors of spermidine biosynthesis, additionally suggessted a possible involvement of polyamines in cytokinin action.     

Effects of inhibitors of spermidine and spermine synthesis on polyamine concentrations and growth of transformed mouse fibroblasts

1. A number of compounds known to inhibit polyamine biosynthesis at various steps in the biosynthetic pathway were tested for their ability to inhibit growth and decrease polyamine concentrations in virally transformed mouse fibroblasts (SV-3T3 cells). 2. Virtually complete inhibition of growth was produced by the inhibitors of ornithine decarboxylase α-methylornithine and α-difluoromethylornithine and by the inhibitors of S-adenosylmethionine decarboxylase 1,1′-[(methylethanediylidene)dinitrilo]diguanidine and 1,1′-[(methylethanediylidene)dinitrilo]bis-(3-aminoguanidine). The former inhibitors decreased putrescine and spermidine contents in the cells to very low values, whereas the latter substantially increased putrescine but decreased spermidine concentrations. The inhibitory effects of all of these inhibitors on cell growth could be prevented by the addition of spermidine, suggesting that spermidine depletion is the underlying cause of their inhibition of growth. 3. α-Difluoromethylornithine, which is an irreversible inhibitor of ornithine decarboxylase, was a more potent inhibitor of growth and polyamine production (depleting spermidine almost completely and spermine significantly) than α-methylornithine, which is a competitive inhibitor. This was not the case with the inhibitors of S-adenosylmethionine decarboxylase where 1,1′-[(methylethanediylidene)dinitrilo]diguanidine, a reversible inhibitor, was more active than 1,1′-[(methylethanediylidene)dinitrilo]bis-(3-aminoguanidine), an irreversible inhibitor. It is suggested that this effect may be due to the lesser uptake and/or greater chemical reactivity of the latter compound. 4. Various nucleoside derivatives of S-adenosylhomocysteine that inhibited spermidine synthase in vitro did not have significant inhibitory action against polyamine accumulation in the cell. These compounds, which included S-adenosylhomocysteine sulphone, decarboxylated S-adenosylhomocysteine sulphone, decarboxylated S-adenosylhomocysteine sulphoxide and S-adenosyl-4-thio-butyric acid sulphone did not inhibit cell growth or polyamine content until cytotoxic concentrations were added. 5. 5′-Methylthioadenosine, 5′-isobutylthioadenosine and 5′-methylthiotubercidin, which inhibit aminopropyltransferase activity in vitro, all inhibited cell growth and decreased spermidine content. Although these compounds were most active against spermine synthase in vitro, they acted in the cell primarily to decrease spermidine content. Cell growth could not be restored to normal values by addition of spermidine, suggesting that these nucleosides have another inhibitory action towards cellular proliferation. 6. 5′-Methylthioadenosine and 5′-isobutylthioadenosine are degraded by a phosphorylase present in SV3T3 cells, yielding 5-methylthioribose-1-phosphate and 5-isobutylthioribose-1-phosphate respectively, and adenine. This degradation appears to decrease the inhibitory action towards cell growth, suggesting that the nucleosides themselves are exerting the inhibitory action. 5′-Methylthiotubercidin, which is not a substrate for the phosphorylase and is a competitive inhibitor of it, was the most active of these nucleosides in inhibiting cell growth and spermidine content. 5′-Methylthiotubercidin and α-difluoromethylornithine had additive effects on retarding cell growth, but not on cellular spermine accumulation, also suggesting that the primary growth-inhibiting action of the nucleoside was not on polyamine production. 7. These results support the concept that 5′-methylthioadenosine phosphorylase plays an important role in permitting cell growth to continue by preventing the build-up of inhibitory intracellular concentrations of 5′-methylthioadenosine.     

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.     

Rootstocks improve cucumber photosynthesis through nitrogen metabolism regulation under salt stress

We examined the growth, photosynthetic parameters, initial and total ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity, the relative expression of rbcL, rbcS, and rca gene, and nitrogen metabolism of cucumber (Cucumis sativus L. cv. Jinchun No.2, CS) plants grafted onto figleaf gourd (Cucurbita ficifolia Bouché, CF) and pumpkin (Cucurbita moschata Duch. cv. Chaojiquanwang, CM) rootstocks. Growth inhibition under salt stress (90 mM NaCl) was characterized by the irreversible inhibition of CO₂ assimilation in the cucumber plants grafted onto cucumber rootstocks (CS/CS). In contrast, this effect was significantly alleviated by grafting the cucumber plants onto the CF and CM roots (CS/CF, CS/CM). Under NaCl stress, the CS/CF and CS/CM plants exhibited higher photosynthetic activity, higher initial and total Rubisco activity, and higher Rubisco-related gene expression than the CS/CS plants. Salinity resulted in a lesser increase in nitrate content and decrease in free amino acid content in the CS/CF and the CS/CM plants compared with the CS/CS plants. Accordingly, the activity of nitrate reductase, glutamine synthetase, and glutamate synthase decreased significantly, especially in the CS/CS plants. These results suggest that grafting cucumber plants onto salt-tolerant rootstocks enhances Rubisco activity and the expression of Rubisco-related genes by effectively accelerating nitrate transformation into amino acids under NaCl stress, thereby improving the photosynthetic performance of cucumber leaves.     

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.     

24-Epibrassinolide regulates carbohydrate metabolism and increases polyamine content in cucumber exposed to Ca(NO3)2 stress

Key message

This study focuses on the impact of carbohydrate metabolism and endogenous polyamines levels in leaves of cucumber seedlings under salt stress by exogenous BRs.

Abstract

The effects of 24-epibrassinolide (EBL) on carbohydrate metabolism and endogenous content of polyamines were investigated in cucumber seedlings (Cucumis sativus L. cv. Jinyou No. 4) exposed to salinity stress [80 mM Ca(NO₃)₂]. Spraying of exogenous EBL partially enhanced the enzyme activities of sucrose phosphate synthase, sucrose synthase and acid invertase; thus, raising the level of sucrose, fructose and total soluble sugars. The amylase activity was also increased by EBL, companied by the rising of sucrose level. These results indicated that EBL improved the carbohydrate metabolism of cucumber under Ca(NO₃)₂ stress. Moreover, EBL raised the levels of soluble conjugated and insoluble bound polyamines while lowered the free polyamines content, particularly putrescine. Our experiment demonstrated that exogenous EBL elevated stability of cellular membrane and positively improve the carbohydrate metabolism in cucumber growing under Ca(NO₃)₂ stress.     

The Use of Olfactory and Visual Cues in Host Choice by the Capsid Bugs Lygus rugulipennis Poppius and Liocoris tripustulatus Fabricius

Lygus rugulipennis Poppius and Liocoris tripustulatus Fabricius (Heteroptera: Miridae) are pests of glasshouse cucumber and sweet pepper crops respectively. L. rugulipennis has a wide range of foodplants, but L. tripustulatus is specialised with very few food plants. We report behavioural assessments to investigate whether either species exhibits a preference for salad over wild hosts, and whether the role of olfaction and vision in response to cues from host plants can be distinguished. Olfactory responses to leaves were tested in choice chambers. L. rugulipennis was presented nettle (wild host) and a salad leaf of cucumber or sweet pepper, where the salad leaves had higher nitrogen content. L. tripustulatus was tested with nettle and sweet pepper of two different nitrogen contents. Female L. rugulipennis spent more time on the cucumber salad host, and chose it first most often, but males showed no preference. Neither sex discriminated between sweet pepper or nettle leaves, but males made more first contacts with sweet pepper. Neither sex of L. tripustulatus discriminated between sweet pepper and nettle leaves when the sweet pepper had higher nitrogen. When the plant species contained equivalent nitrogen both sexes spent more time on nettle. There was no difference in first choice made by either sex. When visual stimuli were available, and leaves had equivalent nitrogen, L. rugulipennis showed no preference and L. tripustulatus preferred nettle leaves. We conclude that the generalist L. rugulipennis has the ability to use remote olfactory cues for host choice whereas the specialist L. tripustulatus relies mainly on contact chemosensory and gustatory cues.     

Reversal of the growth inhibitory effect of α-difluoromethylornithine by putrescine but not by other divalent cations

Summary Treatment with -difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase (ODC), depletes the putrescine and spermidine content, and reduces the growth rate of Ehrlich ascites tumor cells.The addition of putrescine, which is the immediate precursor of spermidine, promptly replenished the intracellular putrescine and spermidine pools and completely reversed the antiproliferative effect of DFMO. A sequential accumulation of spermine, spermidine and putrescine was observed.1,3-diaminopropane, a lower homolog of putrescine, did not reverse the antiproliferative effect of DFMO, despite its structural similarity and identical positive charge. By inhibiting remaining ODC activity, resistant to 5 mM DFMO, and possibly by inhibiting spermine synthase activity, 1,3-diaminopropane produced a further decrease in total polyamine content by reducing the spermine content.Mg²⁺, which can replace putrescine in many in vitro reactions, completely lacked the capacity to reverse the antiproliferative effect of putrescine and spermidine deficiency.Abbreviations DFMO -difluoromethylornithine - ODC ornithine decarbxylase     

Site-Directed Mutations of the Gatekeeping Loop Region Affect the Activity of Escherichia coli Spermidine Synthase

Spermidine synthase catalyzes the production of spermidine from putrescine and decarboxylated S-adenosylmethionine (dcSAM), and plays a crucial role in cell proliferation and differentiation. The gatekeeping loop identified in the structure of spermidine synthase was predicted to contain residues important for substrate binding, but its correlation with enzyme catalysis has not been fully understood. In this study, recombinant Escherichia coli spermidine synthase (EcSPDS) was produced and its enzyme kinetics was characterized. Site-directed mutants of EcSPDS were obtained to demonstrate the importance of the amino acid residues in the gatekeeping loop. Substitution of Asp158 and Asp161 with alanine completely abolished EcSPDS activity, suggesting that these residues are absolutely required for substrate interaction. Reduction in enzyme activity was observed in the C159A, T160A, and P165Q variants, indicating that hydrophobic interactions contributed by Cys159, Thr160, and Pro165 are important for enzyme catalysis as well. On the other hand, replacement of Pro162 and Ile163 had no influence on EcSDPS activity. These results indicate that residues in the gatekeeping loop of spermidine synthase are indispensable for the catalytic reaction of EcSPDS. To the best of our knowledge, this is the first functional study on the gatekeeping loop of EcSPDS by site-directed mutagenesis.     

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.