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At least 595 records · Page 33Linked to original sources
PubMed2001

Purification, crystallization and preliminary X-ray diffraction analysis of the fructose-1,6-/sedoheptulose-1,7-bisphosphatase of Synechococcus PCC 7942.

Fructose-1,6-/sedoheptulose-1,7-bisphosphatase of Synechococcus PCC 7942, overexpressed from Escherichia coli, has been purified and crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitant. The crystals were monoclinic, with unit-cell parameters a = 80.1, b = 84.2, c = 104.3 A, beta = 101.7 degrees. They belonged to space group P2(1) and diffracted to at least 2.2 A resolution. The calculated V(M) value, based on a tetramer in the asymmetric unit, was 2.2 A(3) Da(-1).

Crystallization↗
PubMed2001

Crystallization and preliminary X-ray diffraction analysis of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase of Synechococcus PCC 7942.

The NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Synechococcus PCC 7942 was crystallized in two different forms by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitant. Form I crystals were hexagonal, space group P6(5) or P6(1), with unit-cell parameters a = b = 91.1, c = 428.6 A, gamma = 120 degrees. Form II crystals were monoclinic, space group C2, with unit-cell parameters a = 152.3, b = 80.9, c = 213.6 A, beta = 103.1 degrees. Native data were collected from a frozen crystal of form I to a resolution of 2.8 A using synchrotron radiation at SPring-8, whereas form II crystals were easily damaged by radiation at room temperature and increased mosaicity in cryoprotectant solutions. A molecular-replacement solution of the form I crystal was obtained in space group P6(5) using the program AMoRe and the structure of the NAD-dependent GAPDH from Bacillus stearothermophilus.

Crystallization↗
PubMed2001

Crystallization and preliminary X-ray diffraction analysis of the light-harvesting protein phycocyanin from the thermophilic cyanobacterium Synechococcus elongatus.

The crystallization and preliminary crystallographic study of phycocyanin from the thermophilic cyanobacterium Synechococcus elongatus is reported. Phycocyanin is composed of alpha- and beta-subunits consisting of 162 and 172 amino-acid residues, respectively. These associate to form an alphabeta heterodimer, which further associates to give a ring-shaped trimer (alphabeta)(3). Two trimers bind head-to-head to form a hexamer (alphabeta)(6). Phycocyanin crystals have been obtained by the sitting-drop vapour-diffusion method with a precipitant solution containing 30%(w/v) PEG 4000 and 100 mM MES pH 7.5-8.0. Using synchrotron radiation, the crystals diffract to 2.0 A resolution. They belong to the trigonal space group R32, with unit-cell parameters a = b = 186.75 (3), c = 59.75 (4) A, alpha = beta = 90, gamma = 120 degrees. Assuming that the crystallographic triad is identical to the threefold axis of the hexamer and with three (alphabeta)(6) molecules in a unit cell, the calculated molar volume (V(M)) is 2.64 A(3) Da(-1). This value corresponds to a solvent content of approximately 53%, with one alphabeta heterodimer occupying the asymmetric unit.

Crystallization↗
PubMed2001

Crystallization and preliminary X-ray diffraction studies of catalase-peroxidase from Synechococcus PCC 7942.

The recombinant catalase-peroxidase of Synechococcus PCC 7942 overexpressed in Escherichia coli was purified and crystallized by the hanging-drop vapour-diffusion method using sodium formate as a precipitant. The crystals belonged to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 109.3, c = 202.0 A. The calculated V(M) value based on a dimer in the asymmetric unit was 1.9 A(3) Da(-1). A native data set was collected to 2.3 A resolution from a frozen crystal using synchrotron radiation at SPring-8.

Bacterial Proteins↗
PubMed1993

Structure determination and refinement of ribulose 1,5-bisphosphate carboxylase/oxygenase from Synechococcus PCC6301.

The structure of an activated quaternary complex of ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) from Synechococcus PCC6301 has been solved by molecular replacement. The protein crystallizes in an orthorhombic P2(1)2(1)2(1) unit cell with a complete L(8)S(8) complex consisting of 4608 residues (37 680 non-hydrogen atoms) in the asymmetric unit. Data were collected both on film and image plate using synchrotron radiation; there were 218 276 unique reflections in the final 2.2 A data set. The eightfold non-crystallographic symmetry could be used both to improve map quality and to reduce the computing requirements of refinement. The coordinates were refined using strict non-crystallographic symmetry constraints. The stereochemistry of the final model is good, and the model has an R value of 20.0% for the reflections between 7 and 2.2 A.

Journal Article↗
PubMed1999

Crystallization and preliminary X-ray analysis of plastocyanin from cyanobacterium Synechococcus sp. PCC 7942.

A plastocyanin from the cyanobacterium Synechococcus sp. PCC 7942 has been crystallized in two different forms by hanging-drop vapour diffusion with ammonium sulfate as precipitant. Form I is hexagonal, space group P61 or P65, with unit-cell dimensions a = b = 34.62 and c = 107.22 A. Form II is tetragonal, space group P41 or P43, with unit-cell dimensions a = b = 43.05 and c = 56.94 A. Form I crystals diffract to 2.5 A using graphite-monochromated Cu Kalpha radiation from a Rigaku RU-300 rotating-anode generator operated at 40 kV and 100 mA. Form II crystals diffract to 1.9 A using synchrotron radiation at beamline BL6A of the Photon Factory (KEK). Molecular-replacement calculations using the structure of plastocyanin from Ulva pertusa have been performed.

Crystallization↗
PubMed1992

Identification of replication and stability functions in the complete nucleotide sequence of plasmid pUH24 from the cyanobacterium Synechococcus sp. PCC 7942.

The complete nucleotide sequence is presented for pUH24, the small plasmid of Synechococcus sp. PCC 7942. pUH24 consists of 7835bp and has a G + C content of 59%. The distribution of translation start and stop codons in the sequence allows 36 open reading frames that potentially encode polypeptides of 50 or more amino acids. We postulate that eight of these open reading frames are actual coding sequences. A region has been identified, by experiment, that contains two functions, designated pmaA and pmaB, involved in the segregational stability of the plasmid. The minimal region of pUH24 fully capable of supporting autonomous replication consists of a 3.6kb DNA fragment, which is almost entirely occupied by two overlapping genes most likely coding for essential replication proteins (repA and repB).

Amino Acid Sequence↗
PubMed1993

Rubisco but not Rubisco activase is clustered in the carboxysomes of the cyanobacterium Synechococcus sp. PCC 7942: Mud-induced carboxysomeless mutants.

The Mud technology of Groisman and Casadaban was adapted to the cyanobacterium Synechococcus sp. PCC 7942. A new high-CO2-requiring (hcr) mutant, hcr Mu28 was isolated following the integration of the Mud element 89 bp upstream of ORFI, at the 5'-flanking region of the rbc operon, which encodes RuBP carboxylase/oxygenase (Rubisco). The integration involved a 7 bp duplication that formed a direct repeat at the integration site, as previously shown in Escherichia coli. The mutant was devoid of apparent carboxysome bodies, which are considered to be important for the availability of CO2 for Rubisco. Immunolabelling studies demonstrated that Rubisco was distributed throughout hcr Mu28 cells, while in the wild type (WT) and in the carboxysome aberrant mutant hcr O221, Rubisco was markedly associated with the carboxysomes. Rubisco activase, however, was evenly distributed throughout the cytosol of the hcr and WT cells, without any preferential association with the apparent carboxysomes.

Base Sequence↗
PubMed1993

Cloning and characterization of the psaE gene of the cyanobacterium Synechococcus sp. PCC 7002: characterization of a psaE mutant and overproduction of the protein in Escherichia coli.

The psaE gene, encoding a 7.5 kDa peripheral protein of the photosystem I complex, has been cloned and characterized from the cyanobacterium Synechococcus sp. PCC 7002. The gene is transcribed as an abundant monocistronic transcript of approximately 325 nt. The PsaE protein has been overproduced in Escherichia coli, purified to homogeneity, and used to raise polyclonal antibodies. Mutant strains, in which the psaE gene was insertionally inactivated by interposon mutagenesis, were constructed and characterized. Although the PS I complexes of these strains were similar to those of the wild type, the strains grew more slowly under conditions which favour cyclic electron transport and could not grow at all under photoheterotrophic conditions. The results suggest that PsaE plays a role in cyclic electron transport in cyanobacteria.

Amino Acid Sequence↗
PubMed2004

Roles of CmpR, a LysR family transcriptional regulator, in acclimation of the cyanobacterium Synechococcus sp. strain PCC 7942 to low-CO(2) and high-light conditions.

The cmp operon of Synechococcus sp. strain PCC 7942, encoding a high-affinity bicarbonate transporter, is induced under low CO(2) conditions by a LysR family protein CmpR. CmpR was found to be required also for induction of the operon by transfer of the cells from low-light to high-light conditions, indicating involvement of a common mechanism in the high-light- and low-CO(2)-responsive regulation. Expression of the high-light inducible genes psbAII and psbAIII, on the other hand, was found to be induced also by low-CO(2) conditions. A single promoter was responsible for the high-light and low-CO(2) induction of each of psbAII and psbAIII, suggesting involvement of a common regulatory mechanism in the light and CO(2) responses of the psbA genes. CmpR was, however, not required for the induction of psbAII and psbAIII, indicating the presence of multiple mechanisms for induction of genes under high-light and low-CO(2) conditions. The CmpR-deficient mutant nevertheless showed lower levels of the psbAII and psbAIII transcripts than the wild-type strain under all the light and CO(2) conditions examined. Gel shift assays showed that CmpR binds to the enhancer elements of psbAII and psbAIII, through specific interaction with a sequence signature conforming to the binding motif of similar LysR family proteins. These findings showed that CmpR acts as a trans-acting factor that enhances transcription of the photosystem II genes involved in acclimation to high light, revealing a complex network of gene regulation in the cyanobacterium.

Bacterial Proteins↗
PubMed2004

The Synechococcus elongatus P signal transduction protein controls arginine synthesis by complex formation with N-acetyl-L-glutamate kinase.

This communication identifies, for the first time, a receptor protein for signal perception from the P(II) signal transduction protein in the cyanobacterium Synechococcus elongatus. P(II), a phosphoprotein that signals the carbon/nitrogen status of the cells, forms a tight complex with the key enzyme of the arginine biosynthetic pathway, N-acetylglutamate (NAG) kinase. In complex with P(II), the catalytic activity of NAG kinase is strongly enhanced. Complex formation does not require the effector molecules of P(II), 2-oxoglutarate and ATP, but it is highly susceptible to modifications at the phosphorylation site of P(II), Ser-49. Stable complexes were only formed with the non-phosphorylated form of P(II) but not with Ser-49 mutants. In accordance with these data, NAG kinase activity in S. elongatus extracts correlated with the phosphorylation state of P(II), with high NAG kinase activities corresponding to non-phosphorylated P(II) (nitrogen-excess conditions) and low activities to increased levels of P(II) phosphorylation (nitrogen-poor conditions), thus subjecting the key enzyme of arginine biosynthesis to global nitrogen control.

Adenosine Triphosphate↗
PubMed1979

Purification and primary structure of cytochrome c-552 from the cyanobacterium, Synechococcus PCC 6312.

Cytochrome c-552 (soluble 'cytochrome f') from the unicellular cyanobacterium Synechococcus PCC 6312 (ATCC 27167) was purified and the primary structure determined. The proposed sequence consists of one polypeptide chain of 87 residues. The sequence was determined by a combination of chemical and enzymatic cleavage, manual and automatic sequencing and mass spectroscopy. This is the first amino acid sequence of this cytochrome from a unicellular cyanobacterium to be determined in a study of the variation in primary structure between phylogenetically distant cyanobacteria. The sequence is compared to the primary structures of the cytochrome from filamentous cyanobacteria and from eukaryotic algae. The significance of these sequence comparisons to the current hypotheses concerning the origin of eukaryotic cells and their chloroplasts is discussed.

Amino Acid Sequence↗
PubMed1983

Isolation, purification and characterization of the ATPase complex from the thermophilic cyanobacterium Synechococcus 6716.

The ATPase complex is isolated and purified from membrane vesicles of the thermophilic cyanobacterium Synechococcus 6716 by octyl glucoside and cholic acid by a modification of the procedure for its extraction from spinach chloroplasts. The complex is purified by differential centrifugation and ammonium sulfate precipitation and by gel filtration on Sepharose 6B. The purified fraction, without any phycocyanin contamination, shows ATP hydrolysis activity and Pi/ATP exchange activity of 1564 and 350 nmol X min-1 X mg protein-1, respectively. N,N'-Dicyclohexylcarbodiimide inhibits the ATP hydrolysis activity of this purified fraction. On polyacrylamide gels most typical F1 ATPase polypeptides are identified, but the low-molecular weight polypeptides visible cannot be ascribed to the F0 part of the complex with certainty; non-identified bands around 30 kDa are also present.

Adenosine Triphosphatases↗
PubMed1983

Characterization of reconstituted ATPase complex proteoliposomes prepared from the thermophilic cyanobacterium Synechococcus 6716.

The preparation and some properties are described of proteoliposomes consisting of the ATPase complex and lipids from the thermophilic cyanobacterium Synechococcus 6716. In the proteoliposomes (about 200 nm in diameter) only a low amount of protein can be incorporated (protein/lipid ratio of 0.01 w/w) and they show very few protein particles on freeze-fracture replicas. The octyl glucoside and cholate dialysis method of reconstitution yielded stable proteoliposomes with a relatively low proton permeability. ATP hydrolysis and 32Pi/ATP exchange activities were about 400 and 120 nmol X min-1 X mg protein-1, respectively; the former was strongly stimulated by an uncoupler. ATP hydrolysis induces membrane energization as monitored by membrane-potential- and surface-potential-indicating probes and by different pH indicators trapped inside the vesicles. The probes used were a membrane-bound fluorescent aminoacridine, which monitors surface charge-density changes, the native carotenoids and added oxonol VI for monitoring electrical potential in the membrane and the pH indicators neutral red and cresol red. The different rise kinetics of these probes indicate that proton accumulation upon ATP hydrolysis involves at least two steps: a membrane-localized potential charge and proton transfer followed by a much slower acidification of the bulk intravesicular space. Internal neutral red and cresol red seem to discriminate between proton translocation to the internal interface and bulk space, respectively.

Adenosine Triphosphatases↗
PubMed1984

Proton movements and electric potential generation in reconstituted ATPase proteoliposomes from the thermophilic cyanobacterium Synechococcus 6716.

ATP hydrolysis-induced proton translocation and electric potential generation have been studied in ATPase proteoliposomes by means of various optical probes. The proteoliposomes consisted of reconstituted ATPase complex and native lipid mixture isolated from the thermophilic cyanobacterium Synechococcus 6716 [Van Walraven et al. (1983) Eur. J. Biochem. 137, 101-106]. The native cartenoids and added oxonol VI served as probes for the electric membrane potential generated by the net charge separation (negative outside, positive inside). Their responses, with similar half-times as 9-tetradecylamino-6-chloro-2-methoxyacridine, are sensitive to valinomycin and stimulated by nigericin, as expected. The proton concentrations of extraliposomal and intraliposomal aqueous spaces were monitored by neutral red and cresol red; for internal measurements these pH indicators were trapped inside the vesicles during detergent dialysis. Internal acidification and external alkalinization induced by ATP hydrolysis are inhibited by nigericin and enhanced by valinomycin; at the commonly used higher valinomycin concentrations the neutral red response becomes transient, while the much slower cresol red response is diminished right from its onset. At smaller preset pH gradients both ATP hydrolysis activity and neutral red response are diminished. At increasing MgCl2 concentrations the neutral red responses are slowed down and the cresol red responses are slightly enhanced; this is observed for both internal and external dye responses. Neutral red permeation through the membrane is insignificant under our experimental conditions but is enhanced at temperatures below the lipid-phase transition. In the case of externally added neutral red the non-permeant buffer Hepes is only effective at high MgCl2 concentration, whereas some external cresol red response is visible only at high MgCl2 concentration in the presence of Hepes. The kinetics of the pH indicator and electric potential probe responses clearly distinguish fast interfacial and intra-membrane proton displacements from slow bulk proton equilibration. The data are summarized in a model that supports the importance of localized proton displacements for the primary energy-transducing events.

Adenosine Triphosphatases↗
PubMed1985

Fructose-1,6-bisphosphatase from Synechococcus leopoliensis. Substrate-dependent dimer-tetramer interconversion.

Extracts of Synechococcus leopoliensis (Anacystis nidulans) contain two forms of D-fructose-1,6-bisphosphatase (EC 3.1.3.11) previously designated as forms A and B [Gerbling, K.-P., Steup, M., and Latzko, E. (1984) Arch. Microbiol. 137, 109-114]. Form B, which probably represents the major part of the total extractable fructose-1,6-bisphosphatase activity, has been purified to apparent homogeneity. Gel filtration, non-denaturing polyacrylamide gel electrophoresis, and cross-linking with bis(sulfosuccinimidyl)suberate revealed that the fructose-1,6-bisphosphatase B exists in either a dimeric or in a tetrameric subform, depending upon the absence or presence of fructose-1,6-bisphosphate and Mg2+. The dimer--tetramer interconversion was readily reversible. The results provide evidence for a two-step activation of fructose-1,6-bisphosphatase B involving the reduction of the dimeric subform and the subsequent substrate-dependent conversion of the reduced dimer to a reduced tetramer, which is the only catalytically active state. In contrast to form B, no substrate-dependent interconversion was detected with form A from S. leopoliensis.

Chromatography, Gel↗
PubMed1991

Characterization of glutamate-1-semialdehyde aminotransferase of Synechococcus. Steady-state kinetic analysis.

Synechococcus glutamate-1-semialdehyde aminotransferase was expressed in large amounts in transformed cells of Escherichia coli. The resulting purified enzyme has an absorption spectrum characteristic of B6-containing enzymes and could be converted to the pyridoxal-phosphate form with excess dioxovalerate (O2Val), and back to the pyridoxamine-phosphate form with diaminovalerate (A2Val). Both enzyme forms are similarly active in the conversion of glutamate 1-semialdehyde (GSA) to 5-aminolevulinate (ALev), suggesting that A2Val and O2Val are intermediates. Initial rates of ALev synthesis at various fixed concentrations of GSA followed typical Michaelis-Menten kinetics (Km of GSA for the pyridoxamine-phosphate form of GSA aminotransferase = 12 microM, kcat = 0.23 s-1). In submicromolar amounts A2Val stimulates ALev synthesis, and in a series of concentrations with various fixed concentrations of GSA, gives a family of parallel lines in Lineweaver-Burk plots (Km for A2Val = 1.0 microM). On the other hand, O2Val gives competitive inhibition of the pyridoxamine-phosphate form of GSA-aminotransferase and mixed-type inhibition of the pyridoxal-phosphate form (Ki for O2Val = 1.4 mM). In general the kinetics were typical of ping-pong bi-bi mechanisms in which A2Val is the second substrate (intermediate) and O2Val is an alternative first substrate. There is no compelling evidence that O2Val accepts an amino group at its C5 position resulting in the direct formation of ALev, or the reverse involving the apparent formation of O2Val from ALev. These results are consistent with the hypothesis that the mechanism of GSA aminotransferase mimics that of other aminotransferases and that A2Val is the intermediate.

Chromatography, Ion Exchange↗
PubMed1992

Purification and characterization of the ferredoxin-glutamate synthase from the unicellular cyanobacterium Synechococcus sp. PCC 6301.

Ferredoxin-glutamate synthase from the unicellular cyanobacterium Synechococcus sp. PCC 6301 has been purified using, as main steps, ethanol fractionation in the presence of high ionic strength, ion-exchange chromatography and ferredoxin-Sepharose affinity chromatography. The overall process yielded an homogeneous enzyme with a specific activity of 30 U/mg protein, after a purification of 2800-fold with a recovery of 43%. The molecular mass of the native protein was 156 kDa, as calculated from its Stokes radius (rS, 4.32 nm) and sedimentation coefficient (S20,w, 8.46 S). The size was also estimated by SDS/PAGE as 160 kDa, indicating that the native protein was a monomer. The enzyme exhibited absorption maxima at 279, 370 and 438 nm and a A279/A438 absorbance ratio of 11. One molecule of FMN, but not FAD, was found/molecule native protein. The addition of dithionite resulted in the loss of the absorption peak at 438 nm, which was restored by the addition of 2-oxoglutarate, thus indicating that the prosthetic group is functional in catalysis. Classical hyperbolic kinetics with substrate inhibition was seen for 2-oxoglutarate. The Km values determined for glutamine and ferredoxin were 0.7 mM and 7 microM, respectively, and the apparent Km for 2-oxoglutarate was estimated to be 1.7 mM. Azaserine and 6-diazo-5-oxo-L-norleucine were potent inhibitors of the activity, while pyridoxal 5-phosphate, known to react with Lys residues, partially inactivated the enzyme. This ferredoxin-dependent glutamate synthase is, as far as we know, the first purified from prokaryotic organisms and resembles its counterpart from chloroplasts, suggesting that cyanobacterial glutamate synthase may have been the ancestor of ferredoxin-glutamate synthase in plants.

Amino Acid Oxidoreductases↗