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Query: UMLS:C1832526 (PCC)
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In order to confirm the amino acid sequence predicted from the nucleotide sequence of cDNA and also to elucidate the intracellular localization and molecular evolution, human liver alanine-glyoxylate transaminase 1 (AGT1) was purified and subjected to partial amino acid sequence determination, with special attention to posttranslational modification. The enzyme was purified to homogeneity from the 10,000 x g supernatant of human liver homogenate. The purified enzyme showed only a single protein band at about 43 kDa on SDS-PAGE, indicating that it is a homodimer of two identical subunits, because the native enzyme has a molecular mass of about 80 kDa. Both the amino- and carboxyl-terminal peptides of the enzyme were isolated from a cyanogen bromide digest of the S-carboxyl-methylated protein and subjected to amino acid sequence determination. The alpha-amino group of the amino-terminal peptide was shown to be blocked by an acetyl group. The carboxyl-terminal sequence contained a putative N-glycosylation sequence (-Asn-Ala-Thr-), the only one present in the whole molecule, but this sequence was normally determined, indicating that the enzyme is not N-glycosylated. Purdue et al. [J. Cell Biol. 111, 2341-2351 (1990)] have reported that Pro-11, Gly-170, and Ile-340 in normal human AGT1 were replaced by Leu, Arg, and Met, respectively, in a patient with primary hyperoxaluria type 1. We confirmed that residue-11 was Pro. Both the amino- and carboxyl-terminal sequences of the enzyme showed extensive similarity with those of rat liver mitochondrial serine-pyruvate aminotransferase and the small chain of hydrogenase from a thermophilic unicellular cyanobacterium, Synechococcus PCC 6716.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Purification and amino- and carboxyl-terminal amino acid sequences of alanine-glyoxylate transaminase 1 from human liver. 779 68

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

Photosystem II is a reaction center protein complex located in photosynthetic membranes of plants, algae, and cyanobacteria. Using light energy, photosystem II catalyzes the oxidation of water and the reduction of plastoquinone, resulting in the release of molecular oxygen. A key component of photosystem II is cytochrome b559, a membrane-embedded heme protein with an unknown function. The cytochrome is unusual in that a heme links two separate polypeptide subunits, alpha and beta, either as a heterodimer (alphabeta) or as two homodimers (alpha2 and beta2). To determine the structural organization of cytochrome b559 in the membrane, we used site-directed mutagenesis to fuse the coding regions of the two respective genes in the cyanobacterium Synechocystis sp. PCC 6803. In this construction, the C terminus of the alpha subunit (9 kDa) is attached to the N terminus of the beta subunit (5 kDa) to form a 14-kDa alphabeta fusion protein that is predicted to have two membrane-spanning alpha-helices with antiparallel orientations. Cells containing the alphabeta fusion protein grow photoautotrophically and assemble functional photosystem II complexes. Optical spectroscopy shows that the alphabeta fusion protein binds heme and is incorporated into photosystem II. These data support a structural model of cytochrome b559 in which one heme is coordinated to an alpha2 homodimer and a second heme is coordinated to a beta2 homodimer. In this model, each photosystem II complex contains two cytochrome b559 hemes, with the alpha2 heme located near the stromal side of the membrane and the beta2 heme located near the lumenal side.
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PMID:Structural model of cytochrome b559 in photosystem II based on a mutant with genetically fused subunits. 939 Nov 72

Cyanophycin (multi-L-arginyl-poly-L-aspartate), a water-insoluble reserve polymer of cyanobacteria, is a product of nonribosomal peptide synthesis. The purification of cyanophycin synthetase of the cyanobacterium Anabaena variabilis is described. In sodium dodecylsulfate/polyacrylamide gel electrophoresis, the enzyme preparation shows one band with an apparent molecular mass of 100 kDa. The native enzyme has an apparent molecular mass of approximately 230 kDa, as determined by size-exclusion chromatography, suggesting that the active form is a homodimer. During catalysis, ATP is converted to ADP. The gene coding for cyanophycin synthetase has been identified in the sequenced genome of Synechocystis sp. PCC 6803. The C-terminal 60% of the deduced amino acid sequence of cyanophycin synthetase show sequence similarity to enzymes of the superfamily of ligases involved in the biosynthesis of murein and of folyl-poly(gamma-glutamate). Cells of Escherichia coli harbouring the gene on a plasmid express active synthetase and accumulate cyanophycin-like material. The results prove that a single enzyme catalyzes the de novo synthesis of cyanophycin.
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PMID:Molecular characterization of cyanophycin synthetase, the enzyme catalyzing the biosynthesis of the cyanobacterial reserve material multi-L-arginyl-poly-L-aspartate (cyanophycin). 965 8

Three open reading frames of Synechocystis sp. PCC 6803 encoding a domain homologous with the cAMP binding domain of bacterial cAMP receptor protein were analyzed. These three open reading frames, sll1371, sll1924, and slr0593, which were named sycrp1, sycrp2, and sypk, respectively, were expressed in Escherichia coli as His-tagged or glutathione S-transferase fusion proteins and purified, and their biochemical properties were investigated. The results obtained for equilibrium dialysis measurements using these recombinant proteins suggest that SYCRP1 and SYPK show a binding affinity for cAMP while SYCRP2 does not. The dissociation constant of His-tagged SYCRP1 for cAMP is approximately 3 microM. A cross-linking experiment using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide revealed that His-tagged SYCRP1 forms a homodimer, and the presence or absence of cAMP does not affect the formation of the homodimer. The amino acid sequence reveals that SYCRP1 has a domain similar to the DNA binding domain of bacterial cAMP receptor protein in the COOH-terminal region. Consistent with this, His-tagged SYCRP1 forms a complex with DNA that contains the consensus sequence for E. coli cAMP receptor protein in the presence of cAMP. These results strongly suggest that SYCRP1 is a novel cAMP receptor protein.
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PMID:Identification and characterization of a novel cAMP receptor protein in the cyanobacterium Synechocystis sp. PCC 6803. 1069 19

In Azotobacter vinelandii and Escherichia coli NifS or NifS-like proteins are involved in FeS protein assembly by mobilizing sulfur from free cysteine. This sulfur together with Fe(2+) is then incorporated into apo-FeS proteins to form an FeS center. A different activity termed C-DES [for cyst(e)ine desulfurylase] was recently isolated from the cyanobacterium Synechocystis PCC 6714 which also mobilized sulfur and which was able to incorporate the FeS center into apoferredoxin. In the genome of the cyanobacterium Synechocystis PCC 6803, there are three open reading frames (orfs) that are similar to NifS and one that is similar to C-DES, indicating that this bacterium might contain both activities, NifS and C-DES. One orf from Synechocystis PCC 6803 encoding a NifS-like protein, slr0387, was overexpressed in E. coli and purified. The molecular mass of the recombinant protein was determined to be about 82 kDa, indicating that it is a homodimer. The absorption spectrum was typical for PLP-containing proteins with an absorption maximum at 390 nm at pH 9.0 and at 425 nm at pH 6.5. The pH dependence of the absorption spectrum correlated with enzyme activity. Maximal activity measured as sulfide production was observed between pH 8.5 and 10. The activity decreased at lower pH values and was undetectable at pH 5.5. pH-dependent changes in the absorption spectrum and activity were attributed to protonation of the Schiff base formed by a lysine side chain and the PLP cofactor. Studies on substrate specificity demonstrated that cysteine derivatives other than cysteine methyl ester and cysteine-sulfinic acid could not serve as substrates for this enzyme. In particular, cystine was not a substrate for the Synechocystis NifS-like protein, whereas it is the best substrate for C-DES. In the presence of Fe(2+), cysteine, and a reductant, the NifS-like protein was able to produce holoferredoxin from apoferredoxin. The implications of two different activities for FeS center biosynthesis in Synechocystis are discussed.
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PMID:Role of a NifS-like protein from the cyanobacterium Synechocystis PCC 6803 in the maturation of FeS proteins. 1072 36

The gene cyaB1 from the cyanobacterium Anabaena sp. PCC 7120 codes for a protein consisting of two N-terminal GAF domains (GAF-A and GAF-B), a PAS domain and a class III adenylyl cyclase catalytic domain. The catalytic domain is active as a homodimer, as demonstrated by reconstitution from complementary inactive point mutants. The specific activity of the holoenyzme increased exponentially with time because the product cAMP activated dose dependently and nucleotide specifically (half-maximally at 1 microM), identifying cAMP as a novel GAF domain ligand. Using point mutants of either the GAF-A or GAF-B domain revealed that cAMP activated via the GAF-B domain. We replaced the cyanobacterial GAF domain ensemble in cyaB1 with the tandem GAF-A/GAF-B assemblage from the rat cGMP-stimulated phosphodiesterase type 2, and converted cyaB1 to a cGMP-stimulated adenylyl cyclase. This demonstrated the functional conservation of the GAF domain ensemble since the divergence of bacterial and eukaryotic lineages >2 billion years ago. In cyanobacteria, cyaB1 may act as a cAMP switch to stabilize committed developmental decisions.
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PMID:A GAF-domain-regulated adenylyl cyclase from Anabaena is a self-activating cAMP switch. 1211 May 80

HetR plays a key role in regulation of heterocyst differentiation. When the Cys-48 residue of the HetR from Anabaena sp. PCC 7120 was replaced with an Ala residue, the mutant HetR (HetR(C48A)) could not dimerize, indicating that HetR forms a homodimer through a disulfide bond. The Anabaena strain C48, containing the hetRc48a gene, could not produce HetR homodimer and failed to form heterocyst. We show that HetR is a DNA-binding protein and that its homodimerization is required for the DNA binding. HetR binds the promoter regions of hetR, hepA, and patS, suggesting a direct control of the expression of these genes by HetR. We present evidence that shows that the up-regulation of patS and hetR depends on DNA binding by HetR dimer. The pentapeptide RGSGR, which is present at the C terminus of PatS and blocks heterocyst formation, inhibits the DNA binding of HetR and prevents hetR up-regulation.
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PMID:HetR homodimer is a DNA-binding protein required for heterocyst differentiation, and the DNA-binding activity is inhibited by PatS. 1505 91

The Atx1 copper metallochaperone from Synechocystis PCC 6803, ScAtx1, interacts with two P(1)-type copper ATPases to supply copper proteins within intracellular compartments, avoiding ATPases for other metals en route. Here we report NMR-derived solution structures for ScAtx1. The monomeric apo form has a betaalphabetabetaalpha fold with backbone motions largely restricted to loop 1 containing Cys-12 and Cys-15. The tumbling rate of Cu(I)ScAtx1 (0.1-0.8 mm) implies dimers. Experimental restraints are satisfied by symmetrical dimers with Cys-12 or His-61, but not Cys-15, invading the copper site of the opposing subunit. A full sequence of copper ligands from the cell surface to thylakoid compartments is proposed, considering in vitro homodimer liganding to mimic in vivo liganding in ScAtx1-ATPase heterodimers. A monomeric high resolution structure for Cu(I)ScAtx1, with Cys-12, Cys-15, and His-61 as ligands, is calculated without violations despite the rotational correlation time. (2)J(NH) couplings in the imidazole ring of His-61 establish coordination of N(epsilon2) to copper. His-61 is analogous to Lys-65 in eukaryotic metallochaperones, stabilizing Cu(I)S(2) complexes but by binding Cu(I) rather than compensating charge. Cys-Cys-His ligand sets are an emergent theme in some copper metallochaperones, although not in related Atx1, CopZ, or Hah1. Surface charge (Glu-13) close to the metal-binding site of ScAtx1 is likely to support interaction with complementary surfaces of copper-transporting ATPases (PacS-Arg-11 and CtaA-Lys-14) but to discourage interaction with zinc ATPase ZiaA and so inhibit aberrant formation of copper-ZiaA complexes.
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PMID:Solution structures of a cyanobacterial metallochaperone: insight into an atypical copper-binding motif. 1507 18

Photoreceptor chromoproteins undergo light-induced conformational changes that result in a modulation of protein interaction and enzymatic activity. Bacterial phytochromes such as Cph1 from the cyanobacterium Synechocystis PCC 6803 are light-regulated histidine kinases in which the light signal is transferred from the N-terminal chromophore module to the C-terminal kinase module. In this study, purified recombinant Cph1 was subjected to limited proteolysis using trypsin and endoproteinase Glu-C (V8). Cleavage sites of chromopeptide fragments were determined by MALDI-TOF and micro-HPLC on-line with tandem mass spectrometry in an ion trap mass spectrometer. Trypsin produced three major chromopeptides, termed F1 (S56 to R520), F2 (T64 to R472), and F3 (L81 to R472). F1 was produced only in the far-red absorbing form Pfr within 15 min and remained stable up to >1 h; F2 and F3 were obtained in the red-light absorbing form Pr within ca. 5-10 min. When F1 was photoconverted to Pr in the presence of trypsin, this fragment degraded to F2 and F3 within 1-2 min. On size exclusion chromatography, F1 eluted as a dimer in the Pfr and as a monomer in the Pr form, whereas F2 and F3 behaved always as monomers, irrespective of the light conditions. These and other results are discussed in the context of light-dependent subunit interactions, in which amino acids 473-520 within the PHY domain are required for chromophore-module subunit interaction within the homodimer. V8 proteolysis yielded five major chromopeptides, F4 (T17 to N449), F5 (T17 to E335), F6 (T17 to E323), F7 (unknown sequence), and F8 (tentatively L121 to E323). F6 and F8 were formed in the Pr form, whereas F4, F5, and F7 were preferentially formed in the Pfr form. Three amino acids next to specific cleavage sites, R520, R472, and E323, were altered by site-directed mutagenesis. The mutants were analyzed by UV-vis spectroscopy, size exclusion chromatography, and autophosphorylation. Histidine kinase activity was low in R472A, R520P, and R520A; in all mutants, the ratio of phosphorylation intensity between Pr and Pfr was reduced. Thus, light regulation of autophosphorylation is negatively affected in all mutants. In R472P, E323P, and E323D, the phosphorylation intensity of the Pfr form exceeded that of the wild-type control. This result shows that the histidine kinase activity of Cph1 is actively inhibited by photoconversion into Pfr.
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PMID:Light-induced conformational changes of cyanobacterial phytochrome Cph1 probed by limited proteolysis and autophosphorylation. 1564 69


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