Molecular characterization and homology modeling of spermidine synthase from <Emphasis Type="Italic">Synechococcus</Emphasis> sp. PCC 7942

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.     

Salt stress enhances choline uptake in the halotolerant cyanobacterium Aphanothece halophytica

The uptake of [14C]choline by a suspension of exponential-phase Aphanothece halophytica under various conditions has been studied. Salt stress was found to enhance the uptake of choline. The kinetics of choline transport followed the Michaelis-Menten relationship with apparent K(m) values of 272 and 286 microM, maximum rates of transport (V(max)) of 18 and 37 nmol/min/mg protein for unstressed and salt-stressed cells, respectively. Choline uptake under salt stress was significantly reduced in chloramphenicol-treated cells, suggesting that the activation by salt stress occurred via an inducible transport system. This was corroborated by the existence of the periplasmic choline binding protein, whose content was higher in cells grown under salt-stress condition. Exogenously provided choline significantly increased the growth rate of cells grown under salt stress, although less efficiently than glycine betaine. The presence of 1 mM choline in the growth medium conferred tolerance to high salinity on A. halophytica with the maintenance of high growth up to 1.5 M NaCl. The uptake of choline was Na(+)-dependent, sensitive to various metabolic inhibitors as well as thiol-reactive agents. The results of competition studies suggested that N-methyl on one end of molecule and on the other end either an aldehyde, an alcohol or a neutral group were important features for substrate recognition.     

Degradation of Glycinebetaine by Betaine-Homocysteine Methyltransferase in Aphanothece halophytica: Effect of Salt Downshock and Starvation

We have investigated conditions leading to the degradation of glycinebetaine in Aphanothece halophytica and have shown the activity of betaine-homocysteine methyltransferase (BHMT). The intracellular glycinebetaine level was decreased approximately 50% after 36 h salt downshock from 2.0 m NaCl medium to 0.5 m NaCl medium. A slight additional decrease of glycinebetaine occurred when salt downshock was combined with dark treatment. The omission of carbon and nitrogen sources in the growth medium further decreased intracellular glycinebetaine. The activity of BHMT increased from 0 to 460 nmol h⁻¹mg⁻¹ after 3 h salt downshock. Higher strength of salt downshock resulted in higher activity of the enzyme. Small increase of the enzyme activity was also observed when A. halophytica was deprived of carbon and nitrogen sources in the growth medium. Received: 17 March 2000 / Accepted: 24 April 2000     

Putrescine transport in a cyanobacterium Synechocystis sp. PCC 6803

The transport of putrescine into a moderately salt tolerant cyanobacterium Synechocystis sp. PCC 6803 was characterized by measuring the uptake of radioactively-labeled putrescine. Putrescine transport showed saturation kinetics with an apparent K(m) of 92 +/- 10 microM and V(max) of 0.33 +/- 0.05 nmol/min/mg protein. The transport of putrescine was pH-dependent with highest activity at pH 7.0. Strong inhibition of putrescine transport was caused by spermine and spermidine whereas only slight inhibition was observed by the addition of various amino acids. These results suggest that the transport system in Synechocystis sp. PCC 6803 is highly specific for polyamines. Putrescine transport is energy-dependent as evidenced by the inhibition by various metabolic inhibitors and ionophores. Slow growth was observed in cells grown under salt stress. Addition of low concentration of putrescine could restore growth almost to the level observed in the absence of salt stress. Upshift of the external osmolality generated by either NaCl or sorbitol caused an increased putrescine transport with an optimum 2-fold increase at 20 mosmol/kg. The stimulation of putrescine transport mediated by osmotic upshift was abolished in chloramphenicol-treated cells, suggesting possible involvement of an inducible transport system.     

Biosorption of iron(III) from aqueous solution by dried biomass of Synechocystis sp. PCC 6803

Journal of Applied Phycology - In this study, the dried biomass of Synechocystis sp. PCC 6803 was used as biosorbent for removing Fe(III)) ions from aqueous solution. The effects of exposure time,...     

Temperature dependence of anion transport in the human red blood cell

Arrhenius plots of chloride and bromide transport yield two regions with different activation energies (Ea). Below 15 or 25 degrees C (for Cl- and Br-, respectively), Ea is about 32.5 kcal/mol; above these temperatures, about 22.5 kcal/mol (Brahm, J. (1977) J. Gen. Physiol. 70, 283-306). For the temperature dependence of SO4(2-) transport up to 37 degrees C, no such break could be observed. We were able to show that the temperature coefficient for the rate of SO4(2-) transport is higher than that for the rate of denaturation of the band 3 protein (as measured by NMR) or the destruction of the permeability barrier in the red cell membrane. It was possible, therefore, to extend the range of flux measurements up to 60 degrees C and to show that, even for the slowly permeating SO4(2-) in the Arrhenius plot, there appears a break, which is located somewhere between 30 and 37 degrees C and where Ea changes from 32.5 to 24.1 kcal/mol. At the break, the turnover number is approx. 6.9 ions/band 3 per s. Using 35Cl- -NMR (Falke, Pace and Chan (1984) J. Biol. Chem. 259, 6472-6480), we also determined the temperature dependence of Cl- -binding. We found no significant change over the entire range from 0 to 57 degrees C, regardless of whether the measurements were performed in the absence or presence of competing SO4(2-). We conclude that the enthalpy changes associated with Cl- - or SO4(2-)-binding are negligible as compared to the Ea values observed. It was possible, therefore, to calculate the thermodynamic parameters defined by transition-state theory for the transition of the anion-loaded transport protein to the activated state for Cl-, Br- and SO4(2-) below and above the temperatures at which the breaks in the Arrhenius plots are seen. We found in both regions a high positive activation entropy, resulting in a low free enthalpy of activation. Thus the internal energy required for carrying the complex between anion and transport protein over the rate-limiting energy barrier is largely compensated for by an increase of randomness in the protein and/or its aqueous environment.     

Phosphate sensing in <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803: SphU and the SphS–SphR two-component regulatory system

The Pho regulon is controlled by the histidine kinase-response regulator pair SphS–SphR in many cyanobacteria and up-regulation of the Pho regulon can be monitored by measuring alkaline phosphatase activity. However, the mechanism regulating signal transduction between SphS and SphR has not been described. We have created a cyanobacterial strain allowing the introduction of mutations into the transmitter domain of SphS. Mutations at Thr-167, adjacent to the H motif of SphS, introduce elevated alkaline phosphatase activity in the presence of phosphate and an enhancement of alkaline phosphatase activity, when compared to the control strain, in phosphate-limiting media. SphU acts as a negative regulator of the SphS–SphR system in Synechocystis sp. PCC 6803 and we show that constitutive alkaline phosphatase activity in the absence of SphU requires signal transduction through SphS and SphR. However, constitutive activity in the absence of SphU is severely attenuated in the ΔSphU:SphS-T167N mutant. Our data suggest that Thr-167 contributes to the mechanism underlying regulation by SphU. We have also assembled a deletion mutant system allowing the introduction of mutations into SphR and show that Gly-225 and Trp-236, which are both conserved in SphR from cyanobacteria, are essential for activation of the Pho regulon under phosphate-limiting conditions.     

Actin deficiency induces cofilin phosphorylation: proteome analysis of HeLa cells after beta-actin gene silencing

Actin-binding proteins regulate the dynamic structure and function of actin filaments in the cell. Much is known about how manipulation of the actin-binding proteins affects the structure and function of actin filaments; however, little is known about how manipulation of actin in the cell affects actin-binding proteins. We addressed this question by utilizing two technologies: RNA interference and 2-dimensional gel electrophoresis. We knocked down beta-actin expression in HeLa cells using short interfering RNA and applied 2-DGE to examine alterations in the HeLa cell proteome. We revealed a 2-5 fold increases of four protein spots on 2-D gels and identified these proteins by mass spectrometry. Three of the four proteins were actin-binding proteins, including cofilin, which promotes both disassembly and assembly of actin filaments but becomes inactivated when phosphorylated. Further examination revealed that the cofilin total protein level barely increased, but the phosphorylated cofilin level increased dramatically in HeLa cells after beta-actin siRNA treatment. These results suggest that in response to siRNA-induced beta-actin deficiency HeLa cells inactivate cofilin by phosphorylation rather than down-regulate its protein expression level. This study also demonstrates that the combination of RNA interference and 2-dimensional gel electrophoresis technologies provides a valuable method to study protein interactions in a specific cellular pathway.     

Biocatalytic acylation of carbohydrates with fatty acids from palm fatty acid distillates

Palm fatty acid distillates (PFAD) are by-products of the palm oil refining process. Their use as the source of fatty acids, mainly palmitate, for the biocatalytic synthesis of carbohydrate fatty acid esters was investigated. Esters could be prepared in high yields from unmodified acyl donors and non-activated free fatty acids obtained from PFAD with an immobilized Candida antarctica lipase preparation. Acetone was found as a compatible non-toxic solvent, which gave the highest conversion yields in a heterogeneous reaction system without the complete solubilization of the sugars. Glucose, fructose, and other acyl acceptors could be employed for an ester synthesis with PFAD. The synthesis of glucose palmitate was optimized with regard to the water activity of the reaction mixture, the reaction temperature, and the enzyme concentration. The ester was obtained with 76% yield from glucose and PFAD after reaction for 74 h with 150 U ml⁻¹ immobilized lipase at 40°C in acetone.     

1H, 15N and 13C chemical shift assignments of the BA42 protein of the psychrophilic bacteria Bizionia argentinensis sp. nov

BA42 is a protein belonging to the psychrophilic bacteria Bizionia argentinensis sp. nov. Bioinformatics analysis showed that it presents significant sequence identity with a Pfam A family, DUF 477, found both in eukarya and eubacteria but of unknown function in all these organisms. Here, we report the NMR spectra assignment of this 145 amino acid protein. These data will allow performing NMR structural studies with the aim of using the three-dimensional structure as relevant information in order to determine the function of this family of proteins.