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Query: UMLS:C1832526 (PCC)
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The high potential cytochrome b-559 of intact spinach chloroplasts was photooxidized by red light with a high quantum efficiency and by far-red light with a very low quantum efficiency, when electron flow from water to Photosystem II was inhibited by a carbonyl cyanide phenylhydrazone (FCCP or CCP). Dithiothreitol, which reacts with FCCP or CCCP, reversed the photooxidation of cytochrome b-559 and restored the capability of the chloroplasts to photoreduce CO2 showing that the FCCP/CCCP effects were reversible. The quantum efficiency of cytochrome b-559 photooxidation by red or far-red light in the presence of FCCP was increased by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone which blocks oxidation of reduced plastoquinone by Photosystem I. When the inhibition of water oxidation by FCCP or CCP was decreased by increased light intensities, previously photooxidized cytochrome b-559 was reduced. Red light was much more effective in photoreducing oxidized high potential cytochrome b-559 than far-red light. The red/far-red antagonism in the redox state of cytochrome b-559 is a consequence of the different sensitivity of the cytochrome to red and far-red light and does not indicate that the cytochrome is in the main path of electrons from water to NADP. Rather, cytochrome b-559 acts as a carrier of electrons in a cyclic path around Photosystem II. The redox state of the cytochrome was shifted to the oxidized side when electron transport from water became rate-limiting, while oxidation of water and reduction of plastoquinone resulted in its shifting to the reduced side.
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PMID:Photoreactions of Cytochrome b-559 and cyclic electron flow in photosystem II of intact chloroplasts. 44 98

NADP-dependent isocitrate dehydrogenase activity has been screened in several cyanobacteria grown on different nitrogen sources; in all the strains tested isocitrate dehydrogenase activity levels were similar in cells grown either on ammonium or nitrate. The enzyme from the unicellular cyanobacterium Synechocystis sp. PCC 6803 has been purified to electrophoretic homogeneity by a procedure that includes Reactive-Red-120-agarose affinity chromatography and phenyl-Sepharose chromatography as main steps. The enzyme was purified about 600-fold, with a yield of 38% and a specific activity of 15.7 U/mg protein. The native enzyme (108 kDa) is composed of two identical subunits with an apparent molecular mass of 57 kDa. Synechocystis isocitrate dehydrogenase was absolutely specific for NADP as electron acceptor. Apparent Km values were 125, 59 and 12 microM for Mg2+, D,L-isocitrate and NADP, respectively, using Mg2+ as divalent cation and 4, 5.7 and 6 microM for Mn2+, D,L-isocitrate and NADP, respectively, using Mn2+ as a cofactor. The enzyme was inhibited non-competitively by ADP (Ki, 6.4 mM) and 2-oxoglutarate, (Ki, 6 mM) with respect to isocitrate and in a competitive manner by NADPH (Ki, 0.6 mM). The circular-dichroism spectrum showed a protein with a secondary structure consisting of about 30% alpha-helix and 36% beta-pleated sheet. The enzyme is an acidic protein with an isoelectric point of 4.4 and analysis of the NH2-terminal sequence revealed 45% identity with the same region of Escherichia coli isocitrate dehydrogenase. The aforementioned data indicate that NADP isocitrate dehydrogenase from Synechocystis resembles isocitrate dehydrogenase from prokaryotes and shows similar molecular and structural properties to the well-known E. coli enzyme.
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PMID:Purification and properties of NADP-isocitrate dehydrogenase from the unicellular cyanobacterium Synechocystis sp. PCC 6803. 173 Feb 47

The unicellular cyanobacterium Synechocystis sp. PCC 6803 presents a hexameric NAD-specific glutamate dehydrogenase with a molecular mass of 295 kDa. The enzyme differs from the NADP-glutamate dehydrogenase found in the same strain and is coded by a different gene. NAD-glutamate dehydrogenase shows a high coenzyme specificity, catalyzes preferentially glutamate formation and presents Km values for ammonium, NADH and 2-oxoglutarate of 4.5 mM, 50 microM and 1.8 mM respectively. An animating role for the enzyme is discussed.
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PMID:An NAD-specific glutamate dehydrogenase from cyanobacteria. Identification and properties. 190 12

Structural and chemical properties of a flavodoxin from Anabaena PCC 7119 are described. The first 36 residues of the amino-terminal amino acid sequence have been determined and show extensive homology with flavodoxins isolated from other sources. Anabaena flavodoxin exhibits a net negative change (-3) in the helix-1 segment as found with other cyanobacterial flavodoxins Synechococcus 6301 (Anacystis nidulans) and Nostoc MAC, but in contrast to the net positive charge found in this region in the case of flavodoxins isolated from nitrogen-fixing bacteria (Azotobacter and Klebsiella). The FMN cofactor can be reversibly resolved from the apoprotein by trichloroacetic acid treatment. Apoflavodoxin, thus prepared, binds FMN with a Kd value of 0.1 nM and binds riboflavin with a decreased affinity (Kd = 5 microM) at pH 7.2. The apoprotein is stable in dilute solutions at pH values around 7 but readily denatures at pH 8 as judged from loss in flavin-binding ability and by ultraviolet circular dichroism spectroscopy. Oxidation-reduction potential studies at pH values of 7 and 8 show OX/SQ couples of -195 mV and -255 mV, respectively, and show SQ/HQ couples of -390 mV and -418 mV, respectively. From these data, the binding constant for the FMN semiquinone is calculated to be approx. 5-fold tighter and the binding of the FMN hydroquinone is approx. 10(5)-fold weaker than that of the oxidized FMN to the apoprotein. Anabaena flavodoxin functions as an effective mediator of electron transfer from ferredoxin-NADP(+)-reductase to cytochrome c with a turnover number [4.5-5) x 10(3) min-1); a values similar to that determined for Anabaena ferredoxin. The flavodoxin binds tightly to the reductase with Kd values of 6.4 and 8.5 microM at pH values of 7.0 and 8.0, respectively.
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PMID:Structural and chemical properties of a flavodoxin from Anabaena PCC 7119. 211 31

The gdhA gene of Synechocystis PCC 6803, which encodes an NADP-dependent glutamate dehydrogenase (NADP-GDH), has been cloned by complementation of an Escherichia coli glutamate auxotroph. This gene was found to code for a polypeptide of 428 amino acid residues, whose sequence shows high identity with those of archaebacteria (42-47%), some Gram-positive bacteria (40-44%) and mammals (37%). The minimal fragment of Synechocystis DNA required for complementation (2kb) carries the gdhA gene preceded by an open reading frame (ORF2) encoding a polypeptide of 130 amino acids. ORF2 and gdhA are co-transcribed as a 1.9 kb mRNA, but shorter transcripts including only gdhA were also detected. Two promoter regions were identified upon transcriptional fusion to the cat reporter gene of a promoter probe plasmid. Transcription from the promoter upstream of ORF2 was found to be regulated depending on the growth phase of Synechocystis, in parallel to NADP-GDH activity. This promoter is expressed in Escherichia coli too, in contrast to the second promoter, located between ORF2 and gdhA, which was silent in E. coli and did not respond to the stage of growth in Synechocystis. Disruption of the cyanobacterial gdhA gene with a chloramphenicol resistance cassette yielded a mutant strain totally lacking NADP-GDH activity, demonstrating that this gene is not essential to Synechocystis 6803 under our laboratory conditions.
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PMID:The NADP-glutamate dehydrogenase of the cyanobacterium Synechocystis 6803: cloning, transcriptional analysis and disruption of the gdhA gene. 778 82

NADP(+)-isocitrate dehydrogenase (NADP(+)-IDH) from the dinitrogen-fixing filamentous cyanobacterium Anabaena sp. strain PCC 7120 was purified to homogeneity. The native enzyme is composed of two identical subunits (M(r), 57,000) and cross-reacts with antibodies obtained against the previously purified NADP(+)-IDH from the unicellular cyanobacterium Synechocystis sp. strain PCC 6803. Anabaena NADP(+)-IDH resembles in its physicochemical and kinetic parameters the typical dimeric IDHs from prokaryotes. The gene encoding Anabaena NADP(+)-IDH was cloned by complementation of an Escherichia coli icd mutant with an Anabaena genomic library. The complementing DNA was located on a 6-kb fragment. It encodes an NADP(+)-IDH that has the same mobility as that of Anabaena NADP(+)-IDH on nondenaturing polyacrylamide gels. The icd gene was subcloned and sequenced. Translation of the nucleotide sequence gave a polypeptide of 473 amino acids that showed high sequence similarity to the E. coli enzyme (59% identity) and with IDH1 and IDH2, the two subunits of the heteromultimeric NAD(+)-IDH from Saccharomyces cerevisiae (30 to 35% identity); however, a low level of similarity to NADP(+)-IDHs of eukaryotic origin was found (23% identity). Furthermore, Anabaena NADP(+)-IDH contains a 44-residue amino acid sequence in its central region that is absent in the other IDHs so far sequenced. Attempts to generate icd mutants by insertional mutagenesis were unsuccessful, suggesting an essential role of IDH in Anabaena sp. strain PCC 7120.
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PMID:NADP(+)-isocitrate dehydrogenase from the cyanobacterium Anabaena sp. strain PCC 7120: purification and characterization of the enzyme and cloning, sequencing, and disruption of the icd gene. 816 22

The ADC4 mutant of the cyanobacterium Synechocystis sp. PCC 6803 was studied to determine the structural and functional consequences of the absence of PsaD in photosystem I. Isolated ADC4 membranes were shown to be deficient in ferredoxin-mediated NADP(+) reduction, even though charge separation between P700 and FA/FB occurred with high efficiency. Unlike the wild type, FB became preferentially photoreduced when ADC4 membranes were illuminated at 15 K, and the EPR line shapes were relatively broad. Membrane fragments oriented in two dimensions on thin mylar films showed that the g tensor axes of FA- and FB- were identical in the ADC4 and wild type strains, implying that PsaC is oriented similarly on the reaction center. PsaC and the FA/FB iron-sulfur clusters are lost more readily from the ADC4 membranes after treatment with Triton X-100 or chaotropic agents, implying a stabilizing role for PsaD. The specific role of Lys106 of PsaD, which can be crosslinked to Glu93 of ferredoxin (Lelong et al. (1994) J. Biol. Chem. 269, 10034-10039), was probed by site-directed mutagenesis. Chemical cross-linking and protease treatment experiments did not reveal any drastic alterations in the conformation of the mutant PsaD proteins. The EPR spectra of FA and FB in membranes of the Lys106 mutants were similar to those of the wild type. Membranes of all Lys106 mutants showed wild type rates of flavodoxin reduction and flavodoxin-mediated NADP+ reduction, but had 10-54% decrease in the ferredoxin-mediated NADP+ reduction rates. This implies that Lys106 is a dispensable component of the docking site on the reducing side of photosystem I and an ionic interaction between Lys106 of PsaD and Glu93 of ferredoxin is not essential for electron transfer to ferredoxin. These results demonstrate that PsaD serves distinct roles in modulating the EPR spectral characteristics of FA and FB, in stabilizing PsaC on the reaction center, and in facilitating ferredoxin-mediated NADP+ photoreduction on the reducing side of photosystem I.
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PMID:Mutational analysis of photosystem I polypeptides. Role of PsaD and the lysyl 106 residue in the reductase activity of the photosystem I. 866 33

NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been purified to electrophoretic homogeneity from Synechococcus PCC 7942 cells. The native enzyme had a molecular mass of 160 kDa and consisted of four subunits with a molecular mass of 41 kDa. The activity was 6-fold higher with NADPH than with NADH; the apparent Km values for NADPH and NADH were 62 +/- 4.5 and 420 +/- 10.5 microM respectively. The gene encoding NADP-dependent GAPDH was cloned from the chromosomal DNA of Synechococcus 7942. A 1140 bp open reading frame, encoding an enzyme of 380 amino acid residues (approx.molecular mass of 41.3 kDa) was observed. The deduced amino acid sequence of the gene had a greater sequence similarity to the NADP-dependent and chloroplastic form than to the NAD-dependent and cytosolic form. The Synechococcus 7942 enzyme lacked one of the cysteines involved in the light-dependent regulation of the chloroplast enzymes of higher plants. The recombinant enzyme expressed in Escherichia coli as well as the native enzyme purified from Synechococcus 7942 cells were resistant to 1 mM H2O2.
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PMID:Enzymic and molecular characterization of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Synechococcus PCC 7942: resistance of the enzyme to hydrogen peroxide. 868 18

The crystal structure of the ferredoxin:NADP+ reductase (FNR) from the cyanobacterium Anabaena PCC 7119 has been determined at 2.6 A resolution by multiple isomorphous replacement and refined using 15.0 A to 1.8 A data, collected at 4 degrees C, to an R-factor of 0.172. The model includes 303 residues, the flavin adenine dinucleotide cofactor (FAD), one sulfate ion located at the putative NADP+ binding site and 328 water molecule sites. The structure of Anabaena FNR, including FAD, a network of intrinsic water molecules and a large hydrophobic cavity in the C-terminal domain, resembles that of the spinach enzyme. The major differences concern the additional short alpha-helix (residues 172 to 177 in Anabaena FNR) and residues Arg 100 and Arg 233 which binds NADP+ instead of Lys 116 and Lys 244 in the spinach enzyme. Crystals of a complex of Anabaena FNR with NADP+ were obtained. The model of the complex has been refined using 15 A to 2.25 A X-ray data, collected at -170 degrees C, to an R-factor of 0.186. This model includes 295 residues, FAD, the full NADP+ (with an occupancy of 0.8) and 444 water molecules. The 2'-5' adenine moiety of NADP+ binds to the protein as 2'-phospho-5'-AMP to the spinach FNR. The nicotinamide moiety is turned towards the surface of the protein instead of stacking onto the FAD isoalloxazine ring as would be required for hydride transfer. The model of the complex agrees with previous biochemical studies as residues Arg 100 and Arg 233 are involved in NADP+ binding and residues Arg77, Lys 53 and Lys 294, located on the FAD side of the enzyme, remain free to interact with ferredoxin and flavodoxin, the physiological partners of ferredoxin: NADP reductase.
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PMID:X-ray structure of the ferredoxin:NADP+ reductase from the cyanobacterium Anabaena PCC 7119 at 1.8 A resolution, and crystallographic studies of NADP+ binding at 2.25 A resolution. 889 Sep 10

Glucose-6-phosphate dehydrogenase is a particularly important enzyme in carbon catabolism in the chloroplasts of higher plants and in cyanobacteria. It catalyzes the first reaction in the oxidative pentose phosphate pathway which supplies reduced NADP for a variety of biosynthetic processes. The enzyme is known to be regulated by light. However, the dehydrogenase from plants has been difficult to purify and there is little information on kinetics and mechanism of deactivation. The glucose-6-phosphate dehydrogenase from the heterocystous cyanobacterium, Anabaena sp. PCC 7120, was purified to near homogeneity by chromatography on 2',5'-ADP Sepharose chromatography. The cyanobacterial enzyme apparently has different aggregation states or conformations depending on its concentration in solution and the pH. At a pH of 8.0 and low ionic strength, the enzyme has relatively low activity and exhibits sigmoidal kinetics on binding substrate and cofactor. Activity increases and the enzyme exhibits the more classical hyperbolic kinetics at pH 7.0. At the lower pH, glucose-6-phosphate dehydrogenase is inhibited by catalytic amounts of reduced thioredoxin-1 from Anabaena sp. The second thioredoxin from the cyanobacterium is much less effective, although its inhibitory effect is still greater than that of small molecule reducing agents such as glutathione. Glutamine was reported to stabilize the isolated enzyme, but actually is an activator at pH 8.0. The results suggest that cellular demand for reduced cofactor under nitrogen-fixing conditions overrides the pH-induced deactivation.
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PMID:Glucose-6-phosphate dehydrogenase from the cyanobacterium, Anabaena sp. PCC 7120: purification and kinetics of redox modulation. 890 Apr 2


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