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Query: UMLS:C1832526 (PCC)
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In this work we present the characterization of a hemidiscoidal phycobilisome type of the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. The phycobilisome of this organism contains allophycocyanin, phycocyanin and phycoerythrocyanin, similar to the closely related thermophilic cyanobacterium Mastigocladus laminosus. Intact phycobilisomes exhibit an absorption maximum at 619 nm and two fluorescence maxima at 664 nm and 680 nm, corroborating the presence of a complete energy pathyway along the antenna. Upon dissociation, the phycobiliproteins were released from the phycobilisome. One phycoerythrocyanin, one phycocyanin and three allophycocyanin complexes were isolated by ion-exchange chromatography and characterized by absorption and fluorescence spectroscopy and by SDS/PAGE. The amino-terminal sequences of the polypeptides belonging to the phycoerythrocyanin and phycocyanin families were identical with the derived sequences of their corresponding genes. Partial amino-terminal sequences of the polypeptides belonging to the allophycocyanin family are presented here. Our results show that the phycobiliproteins and linker polypeptides from Anabaena sp. PCC 7120 are similar to the phycobilisome components characterized in other cyanobacteria. The phycobilisome of Anabaena sp. PCC 7120 was extensively analyzed by electron microscopy. It differs from the common hemidiscoidal tricylindrical, six-rod phycobilisome type by a core domain consisting of five core cylinders surrounded by up to eight rods radiating in a hemidiscoidal manner. One rod is linked to each basal core cylinder, whereas the remaining core cylinders bind two rods each. On the basis of the data presented in this work, a revised model for the hemidiscoidal pentacylindrical phycobilisome of Anabaena sp. PCC 7120, M. laminosus and Anabaena variabilis is proposed. This model accounts more accurately for the 'grape' pattern typically exhibited by these phycobilisomes in electron micrographs.
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PMID:Isolation, characterization and electron microscopy analysis of a hemidiscoidal phycobilisome type from the cyanobacterium Anabaena sp. PCC 7120. 866 89

Many filamentous, heterocyst-forming cyanobacteria express a sugar-non-specific nuclease of about 29 kDa that can be detected in DNA-containing SDS-PAGE gels. The nucA gene encoding this nuclease has previously been cloned from Anabaena sp. PCC 7120, sequenced and expressed in Escherichia coli. The NucA protein bears a putative signal peptide close to its N-terminal end and, in Anabaena cultures, is present in both the cells and the extracellular medium. Cell-free extracts of different cyanobacteria producing NucA-like nucleases exhibited an inhibitory activity on NucA. In Anabaena sp. PCC 7120, this inhibition was exerted by protein(s) or protein-containing molecule(s) that were heat resistant. Immediately downstream from the nucA gene, in the complementary strand, we have identified an open reading frame composed of 135 codons, that we have named nuiA, whose expression in E. coli conferred heat-resistant NucA-inhibitory activity to cell-free extracts. The NuiA protein was purified to homogeneity, and purified NuiA inhibited the nuclease activity of NucA. Sequences hybridizing with the nuiA gene have been found in all the tested cyanobacterial strains that express a NucA-like nuclease. Whereas the NucA protein is homologous to endonuclease G from vertebrates and to nucleases from Serratia marcescens and yeast, no protein homologous to NuiA was found in the available databases. Therefore, nuiA represents a novel gene encoding a nuclease inhibitor.
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PMID:The nuiA gene from Anabaena sp. encoding an inhibitor of the NucA sugar-non-specific nuclease. 917 Dec 82

A dihydrolipoamide dehydrogenase (LPD; dihydrolipoamide:NAD oxidoreductase, EC 1.8.1.4.) activity has been detected in the cyanobacterium Synechocystis PCC 6803. The enzyme was isolated from the membraneous fraction after detergent solubilization and shown to be homogenous on the basis of SDS-PAGE and N-terminal sequencing. The isolated enzyme had a specific activity of 75 U (mg protein)(-1) and was shown to be a homodimer with an apparent molecular mass of 104 kDa for the dimer and 55 kDa for the subunits. The enzyme contains 1.75 mol noncovalently bound FAD (mol enzyme)(-1) suggesting that each subunit contains 1 mol FAD and that the FAD is fairly tightly associated with the enzyme. N-terminal sequencing gave a contiguous amino acid sequence of 17 residues and showed that the N-terminus of the LPD from Synechocystis PCC 6803 has significant homologies to other LPDs sequenced so far. Immunoblot experiments indicated that the enzyme is mainly present in the membrane fraction, and immunocytochemical investigations gave evidence that the LPD in Synechocystis PCC 6803 is located in the periplasma space between the cytoplasma membrane and the peptidoglycan layer. This is the first report on an extracellular, membrane-bound LPD in a cyanobacterium.
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PMID:Isolation, partial characterization and localization of a dihydrolipoamide dehydrogenase from the cyanobacterium Synechocystis PCC 6803. 921 12

The phycobilisome (PBS) of Anabaena sp. PCC 7120 was allowed to dissociate into its constituents and the resulting allophycocyanin (AP) fraction was purified. Its reconstitution yielded a complex which according to negative stain electron microscopy and spectral analysis was identical to the native pentacylindrical PBS core domain. Each cylinder of the central tricylindric unit was comprised of four AP (alphabeta)3 disks. Mass analysis using the scanning transmission electron microscope (STEM) showed the presence of 16 AP trimers in the intact reconstitute, which had a total mass of 1966(+/-66) kDa. Composition analysis indicated an AP trimer distribution of (AP-II):(AP-LCM):(AP-B):(AP-I)=6:2:2:6, i.e. an addition of two AP-I and two AP-II complexes compared to a tricylindrical PBS core domain. Therefore, we suggest that each supplementary half-core cylinder found in pentacylindrical AP core domains is comprised of one AP-I and one AP-II trimer, in agreement with the current model. The structural significance of the 127 kDa core membrane linker polypeptide was further investigated by subjecting the AP core reconstitute to mild chymotryptic degradation. After isolation, the digested complex exhibited a tricylindrical appearance while STEM mass analysis confirmed the presence of only 12 AP complexes. Polypeptide analysis by SDS-PAGE and Edman degradation related the half-cylinder loss to cleavage of the Rep4 domain of the core membrane linker polypeptide. On the basis of these data, a general model for the assembly of the three hemidiscoidal PBS types known to date is discussed.
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PMID:Reconstitution, characterisation and mass analysis of the pentacylindrical allophycocyanin core complex from the cyanobacterium Anabaena sp. PCC 7120. 957 Oct 58

Two major proteins, A and B, were isolated and purified from outer membranes of the unicellular cyanobacterium Synechococcus PCC 6301 by gel filtration, anion-exchange chromatography, and preparative SDS-PAGE. Protein A revealed a single-channel conductance of 0.4 nanoSiemens (nS) in 1 M KCl, whereas preparations containing both proteins showed two different conductance maxima of 0.4 and 0.9 nS, suggesting that B also forms pores. The apparent molecular mass of the two closely migrating proteins was determined as 52 kDa, whereas native porin extracts revealed a relative molecular mass of ca. 140 kDa, indicating trimeric pore-forming units. Partial sequences of both proteins were obtained by N-terminal sequencing of tryptic peptides, and the C-terminal amino acid sequences were derived from the complete proteins. These sequences were aligned to protein sequences available in the databases. The results are discussed.
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PMID:Characterization of two pore-forming proteins isolated from the outer membrane of Synechococcus PCC 6301. 960 42

Tetrahydrobiopterin (BH4)-glucoside was identified from Synechococcus sp. PCC 7942 by HPLC analysis and the enzymatic activity of a glycosyltransferase producing the compound from UDP-glucose and BH4. The novel enzyme, named UDP-glucose:BH4 glucosyltransferase, has been purified 846-fold from the cytosolic fraction of Synechococcus sp. PCC 7942 to apparent homogeneity on SDS-PAGE. The native enzyme exists as a monomer having a molecular mass of 39.2 kDa on SDS-PAGE. The enzyme was active over a broad range of pH from 6.5 to 10.5 but most active at pH 10.0. The enzyme required Mn(2+) for maximal activity. Optimum temperature was 42 degrees C. Apparent K(m) values for BH4 and UDP-glucose were determined as 4.3 microM and 188 microM, respectively, and V(max) values were 16.1 and 15.1 pmol min(-1) mg(-1), respectively. The N-terminal amino acid sequence was Thr-Ala-His-Arg-Phe-Lys-Phe-Val-Ser-Thr-Pro-Val-Gly-, sharing high homology with the predicted N-terminal sequence of an unidentified open reading frame slr1166 determined in the genome of Synechocystis sp. PCC 6803, which is known to produce a pteridine glycoside cyanopterin.
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PMID:Purification and characterization of UDP-glucose:tetrahydrobiopterin glucosyltransferase from Synechococcus sp. PCC 7942. 1111 66

A gene argH, encoding argininosuccinate lyase (ASL), has been cloned from a cosmid library of the filamentous cyanobacterium Nostoc sp. strain PCC 73102. The argH open reading frame encodes a protein comprised of 461 amino acids with a calculated molecular mass of 51,349 Da. Protein sequence comparisons reveal significant similarities of the Nostoc PCC 73102 ASL to related proteins from other organisms. In an Escherichia coli delta argH strain, the Nostoc PCC 73102 ASL expressed from a recombinant plasmid could restore the ability to grow on medium without arginine. Moreover, cell extracts show a specific ASL activity of 16.2 nmoles of urea x min(-1) x (mg protein)(-1). Partially purified, His-tagged ASL runs as a 53-kDa protein band in SDS-PAGE and about 215-kDa protein in native-PAGE, suggesting that the native protein is a tetramer.
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PMID:Cloning, characterization, and functional expression in Escherichia coli of argH encoding argininosuccinate lyase in the cyanobacterium Nostoc sp. strain PCC 73102. 1168 60

NAD(P)(+)-reducing hydrogenases have been described to be composed of a diaphorase (HoxFU) and a hydrogenase (HoxYH) moiety. This study presents for the first time experimental evidence that in cyanobacteria, a fifth subunit, HoxE, is part of this bidirectional hydrogenase. HoxE exhibits sequence identities to NuoE of respiratory complex I of Escherichia coli. The subunit composition of the cyanobacterial bidirectional hydrogenase has been investigated. The oxygen labile enzyme complex was purified to close homogeneity under anaerobic conditions from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 6301. The 647-fold and 1290-fold enriched purified enzyme has a specific activity of 46 micromol H(2) evolved (min mg protein)(-1) and 15 micromol H(2) evolved (min mg protein)(-1), respectively. H(2)-evolution of the purified enzyme of S. sp. PCC 6803 is highest at 60 degrees C and pH 6.3. Immunoblot experiments, using a polyclonal anti-HoxE antibody, demonstrate that HoxE co-purifies with the hydrogenase activity in S. sp. PCC 6301. SDS-PAGE gels of the purified enzymes revealed six proteins, which were partially sequenced and identified, besides one nonhydrogenase component, as HoxF, HoxU, HoxY, HoxH and, remarkably, HoxE. The molecular weight of the native protein (375 kDa) indicates a dimeric assembly of the enzyme complex, Hox(EFUYH)(2).
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PMID:HoxE--a subunit specific for the pentameric bidirectional hydrogenase complex (HoxEFUYH) of cyanobacteria. 1203 72

In the absence of exogenous donors, turnover of 10 molar equivalents of H(2)O(2) by wild-type recombinant cytochrome c peroxidase [CCP(MI)] and its W191F mutant at pH 7.0 occurs by oxidation of endogenous donors on the polypeptide. No O(2) evolution was observed with either enzyme on reaction with 10 molar equivalents of H(2)O(2), eliminating catalase-like activity, but O(2) evolution was observed when 100 molar equivalents of H(2)O(2) were added to the enzymes. Protein dimers were observed by SDS-PAGE following H(2)O(2) turnover by the peroxidases, and dimeric forms of CCP(MI) and CCP(W191) were isolated by gel-permeation chromatography. LC-ESI-MS analysis of the tryptic digests of the dimers revealed the previously reported T(6)-T(6) crosslink and a new crosslink between T(6)-T(26), but no T(26)-T(26) crosslink. The crosslinked tryptic peptides contain the exposed tyrosine residues Tyr36, Tyr39 and Tyr42 (T(6)), and Tyr229 and Tyr236 (T(26)). Addition of a spin trap, 2-methyl-2-nitrosopropane (MNP), to the CCP(MI)/H(2)O(2) reaction resulted in MNP labeling of peptides T(6), T(21) (which contains Tyr153) and T(26). MNP labeling of Tyr236 was found by sequencing peptide T(26). MNP labeling did not compete with dimerization of H(2)O(2)-oxidized CCP(W191F), suggesting that dityrosine formation in this mutant is very rapid owing to the high reactivity of radicals formed on T(6). H(2)O(2)-dependent formation of CCP-cytochrome c heterodimers was observed for both CCP(MI) and W191F in the presence of ferricytochrome c, the oxidized form of CCP's donor substrate. Interestingly, no H(2)O(2)-dependent cytochrome crosslinking to the W51F mutant was observed, even though this mutant underwent extensive homocrosslinking. The translocation of oxidizing equivalents from the heme to the surface residues of CCP is discussed in terms of an antioxidant role for CCP.
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PMID:Different pathways of radical translocation in yeast cytochrome c peroxidase and its W191F mutant on reaction with H(2)O(2) suggest an antioxidant role. 1258 60

Light-dependent regulation of a growing number of chloroplast enzymatic activities has been found to occur through the reversible reduction of intra- or intermolecular disulphides by thioredoxins. In cyanobacteria, despite their similarity to chloroplasts, no proteins have hitherto been shown to interact with thioredoxins, and the role of the cyanobacterial ferredoxin/thioredoxin system has remained obscure. By using an immobilized cysteine 35-to-serine site-directed mutant of the Synechocystis sp. PCC 6803 thioredoxin TrxA as bait, we screened the Synechocystis cytosolic and peripheral membrane protein complements for proteins interacting with TrxA. The covalent bond between the isolated target proteins and mutated TrxA was confirmed by nonreducing/reducing two-dimensional SDS/PAGE. Thus, we have identified 18 cytosolic proteins and 8 membrane-associated proteins as candidate thioredoxin substrates. Twenty of these proteins have not previously been associated with thioredoxin-mediated regulation. Phosphoglucomutase, one of the previously uncharacterized thioredoxin-linked enzymes, has not earlier been considered a target for metabolic control through disulphide reduction. In this article, we show that phosphoglucomutase is inhibited under oxidizing conditions and activated by DTT and reduced wild-type TrxA in vitro. The results imply that thioredoxin-mediated redox regulation is as extensive in cyanobacteria as in chloroplasts but that the subjects of regulation are largely different.
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PMID:Thioredoxin-linked processes in cyanobacteria are as numerous as in chloroplasts, but targets are different. 1467 18


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