RESUMO
Spermidine is a ubiquitous polyamine synthesized by spermidine synthase (SPDS) from the substrates, putrescine and decarboxylated S-adenosylmethionine (dcAdoMet). SPDS is generally active as homodimer, but higher oligomerization states have been reported in SPDS from thermophiles, which are less specific to putrescine as the aminoacceptor substrate. Several crystal structures of SPDS have been solved with and without bound substrates and/or products as well as inhibitors. Here, we determined the crystal structure of SPDS from the cyanobacterium Synechococcus (SySPDS) that is a homodimer, which we also observed in solution. Unlike crystal structures reported for bacterial and eukaryotic SPDS with bound ligands, SySPDS structure has not only bound putrescine substrate taken from the expression host, but also spermidine product most probably as a result of an enzymatic reaction. Hence, to the best of our knowledge, this is the first structure reported with both amino ligands in the same structure. Interestingly, the gate-keeping loop is disordered in the putrescine-bound monomer while it is stabilized in the spermidine-bound monomer of the SySPDS dimer. This confirms the gate-keeping loop as the key structural element that prepares the active site upon binding of dcAdoMet for the catalytic reaction of the amine donor and putrescine.
Assuntos
Proteínas de Bactérias/química , Putrescina/química , Espermidina Sintase/química , Synechococcus/enzimologia , Cristalografia por Raios X , Domínios Proteicos , Estrutura Secundária de ProteínaRESUMO
Spermidine synthase (Spds) catalyzes the formation of spermidine by transferring the aminopropyl group from decarboxylated S-adenosylmethionine (dcSAM) to putrescine. The Synechococcus spds gene encoding Spds was expressed in Escherichia coli. The purified recombinant enzyme had a molecular mass of 33 kDa and showed optimal activity at pH 7.5, 37 °C. The enzyme had higher affinity for dcSAM (K m, 20 µM) than for putrescine (K m, 111 µM) and was highly specific towards the diamine putrescine with no activity observed towards longer chain diamines. The three-dimensional structural model for Synechococcus Spds revealed that most of the ligand binding residues in Spds from Synechococcus sp. PCC 7942 are identical to those of human and parasite Spds. Based on the model, the highly conserved acidic residues, Asp89, Asp159 and Asp162, are involved in the binding of substrates putrescine and dcSAM and Pro166 seems to confer substrate specificity towards putrescine.
Assuntos
Putrescina/metabolismo , S-Adenosilmetionina/metabolismo , Espermidina Sintase/química , Espermidina Sintase/metabolismo , Synechococcus/enzimologia , Asparagina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Humanos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Peso Molecular , Prolina/metabolismo , Ligação Proteica , Homologia de Sequência do Ácido Nucleico , Espermidina Sintase/genética , Homologia Estrutural de Proteína , Especificidade por Substrato , Synechococcus/química , Synechococcus/genéticaRESUMO
The Synechococcus sp. PCC 7942 spermidine synthase encoded by spds gene (Synpcc7942_0628) is responsible for spermidine biosynthesis. Two Synechococcus strains, the overexpressing spds (OX-spds) and the spds knockout (Δspds), were constructed and characterized for their growth and photosynthetic efficiency under osmotic stress imposed by sorbitol. The growth of Δspds was completely inhibited when cells were grown in the presence of 400 mM sorbitol. Under the same condition, the OX-spds showed a slightly higher growth than the wild type. The OX-spds under osmotic stress also had a significant increase of spermidine level in conjunction with the up-regulation of the genes involved in spermidine biosynthesis. A higher ratio of spermidine to putrescine, an index for stress tolerance, under osmotic stress was found in the OX-spds strain than in the wild type. Overall results indicated that the spermidine synthase enzyme plays an essential role in the survival of Synechococcus sp. PCC 7942 under osmotic stress.
Assuntos
Proteínas de Bactérias/metabolismo , Espermidina Sintase/metabolismo , Synechococcus/enzimologia , Synechococcus/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Viabilidade Microbiana , Pressão Osmótica , Espermidina Sintase/genética , Synechococcus/química , Synechococcus/genéticaRESUMO
The in vivo function of polyamine binding protein D (PotD) in Synechocystis sp. PCC 6803 for the transport of spermidine was investigated using Synechocystis mutant disrupted in potD gene. The growth rate of potD mutant was similar to that of wild-type when grown in BG11 medium. However, the mutant exhibited severely reduced growth compared to the wild-type when BG11 medium was supplemented with 0.5 mM spermidine. The mutant accumulated a higher spermidine level than the wild-type when grown in the medium with or without spermidine. Transport experiments revealed that the mutant had a reduction in both the uptake and the excretion of spermidine. Moreover, [(14)C]spermidine-loaded wild-type and mutant cells showed a decrease of [(14)C]spermidine excretion when the assay medium contained exogenous spermidine. These data suggest that PotD is involved in both the uptake and the excretion of spermidine in Synechocystis cells.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Poliaminas/metabolismo , Espermidina/metabolismo , Synechocystis/metabolismo , Proteínas de Bactérias/genética , Transporte Biológico , Proteínas de Membrana Transportadoras/genética , Synechocystis/genéticaRESUMO
To investigate the short term effect of ultraviolet (UV) radiations on changes in pigments and polyamine contents, Synechocystis sp. PCC 6803 cells after exposure to UV-radiation were extracted by dimethylformamide and perchloric acid for pigments and polyamines determination, respectively. Cell growth was slightly decreased after 1 h exposure to UV-A and UV-B radiations. UV-C had little effect on cell growth despite the decrease of photosynthetic rate by about 18%. UV-A and UV-B decreased the contents of chlorophyll a and carotenoids whereas UV-C decreased chlorophyll a but had no effect on carotenoids. Spermidine contents were unaffected by UV-A, in contrast to the reduction of 25 and 50% by UV-B and UV-C, respectively. All three types of UV-radiation particularly reduced perchloric acid-insoluble spermidine. Importantly, putrescine and spermine which accounted for less than 1% of intracellular polyamines were increased by about three- to eight-fold by UV-B and UV-C, respectively. The changes in polyamines contents by UV-B and UV-C were consistent with the changes in transcript levels of arginine decarboxylase mRNA, but not with the protein levels. The decrease in the transcripts of adc2 but not adc1 was observed with UV-B and UV-C treatments.
Assuntos
Carboxiliases/biossíntese , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , RNA Mensageiro/análise , Espermidina/análise , Synechocystis/química , Synechocystis/efeitos da radiação , Raios Ultravioleta , Carotenoides/análise , Clorofila/análise , Putrescina/análise , Espermina/análise , Synechocystis/crescimento & desenvolvimentoRESUMO
The effect of UV-B radiation on growth and polyamines content of Synechocystis sp. PCC 6803 subjected to either NaCl or sorbitol stress was investigated. Cells could not grow in the presence of 350 mM NaCl or 500 mM sorbitol under normal white light. However, cells grown in BG11 under osmotic stress imposed by NaCl or sorbitol followed by ultraviolet-B (UV-B) irradiation for 2 h showed higher cell density than those under the same condition but no osmotic stress. The chlorophyll fluorescence parameter (F(v)/F(m)) also showed an apparent decrease upon UV-B irradiation. Intracellular polyamines increased by about 2- and 4-fold in NaCl- and sorbitol-stressed cells, respectively. When these cells were irradiated with UV-B for 1 h, a further 3-fold increase in polyamines content was detected in NaCl-stressed but not sorbitol-stressed cells. Synechocystis cells contained adc1 and adc2 genes encoding arginine decarboxylase (ADC) with only adc1 showing upregulation by NaCl or sorbitol stress. NaCl- or sorbitol-stressed cells contained about 5-fold higher level of adc1 transcript than did the unstressed cells after 1-h irradiation with UV-B, suggesting the protection of adc1 transcript by accumulated polyamines, due to NaCl or sorbitol stress, against UV-B radiation damage. ADC levels as analyzed by Western blot showed upregulation by UV-B in NaCl-stressed but not sorbitol-stressed cells.