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Query: UMLS:C1832526 (PCC)
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We investigated the role of electrostatic charges at positions D72 and K8 in the function and structural stability of cytochrome c6 from Nostoc sp. PCC 7119 (cyt c6). A series of mutant forms was generated to span the possible combinations of charge neutralization (by mutation to alanine) and charge inversion (by mutation to lysine and aspartate, respectively) in these positions. All forms of cyt c6 were functionally characterized by laser flash absorption spectroscopy, and their stability was probed by urea-induced folding equilibrium relaxation experiments and differential scanning calorimetry. Neutralization or inversion of the positive charge at position K8 reduced the efficiency of electron transfer to photosystem I. This effect could not be reversed by compensating for the change in global charge that had been introduced by the mutation, indicating a specific role for K8 in the formation of the electron transfer complex between cyt c6 and photosystem I. Replacement of D72 by asparagine or lysine increased the efficiency of electron transfer to photosystem I, but destabilized the protein. D72 apparently participates in electrostatic interactions that stabilize the structure of cyt c6. The destabilizing effect was reduced when aspartate was replaced by the small amino acid alanine. Complementing the mutation D72A with a charge neutralization or inversion at position K8 led to mutant forms of cyt c6 that were more stable than the wild-type under all tested conditions.
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PMID:Role of the surface charges D72 and K8 in the function and structural stability of the cytochrome c from Nostoc sp. PCC 7119. 1597 38

Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that can fix N2 in differentiated cells called heterocysts. The products of Anabaena open reading frames (ORFs) all1046, all1047, all1284, alr1834 and all2912 were identified as putative elements of a neutral amino acid permease. Anabaena mutants of these ORFs were strongly affected (1-12% of the wild-type activity) in the transport of Pro, Phe, Leu and Gly and also impaired (17-30% of the wild-type activity) in the transport of Ala and Ser. These results identified those ORFs as the nat genes encoding the N-I neutral amino acid permease. According to amino acid sequence homologies, natA (all1046) and natE (all2912) encode ATPases, natC (all1047) and natD (all1284) encode transmembrane proteins, and natB (alr1834) encodes a periplasmic substrate-binding protein of an ABC-type uptake transporter. The natA, natC, natD and natE mutants showed defects in Gln and His uptake that were not observed in the natB mutant suggesting that NatB is not a binding protein for Gln or His. The nat mutants released hydrophobic amino acids to the medium, and amino acid release took place at higher levels in cultures incubated in the absence of combined N than in the presence of nitrate. Alanine was the amino acid released at highest levels, and its release was impaired in a mutant unable to develop heterocysts. The nat mutants were also impaired in diazotrophic growth, with natA, natC, natD and natE mutants showing more severe defects than the natB mutant. Expression of natA and natC, which constitute an operon, natCA, as well as of natB was studied and found to take place in vegetative cells but not in the heterocysts. These results indicate that the N-I permease is necessary for normal growth of Anabaena sp. strain PCC 7120 on N2, and that this permease has a role in the diazotrophic filament specifically in the vegetative cells.
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PMID:ABC-type neutral amino acid permease N-I is required for optimal diazotrophic growth and is repressed in the heterocysts of Anabaena sp. strain PCC 7120. 1613 26

The role played by the residues Leu12 and Lys33 - which are both located at the north hydrophobic patch of plastocyanin - in the interaction of the copper protein with Photosystem I from the cyanobacterium Synechocystis sp. PCC 6803 has been investigated by site-directed mutagenesis. A thermodynamic analysis of PS I reduction by wild-type and mutant plastocyanins has been performed by laser-flash absorption spectroscopy. In all cases, the electron transfer is impaired by mutations, which induce drastic changes in the apparent activation entropy of the overall reaction. Substitution of Leu12 by alanine specifically affects the hydrophobic interactions with PS I, whereas replacement of Lys33 by glutamate not only induces local electrostatic changes, but also alters the hydrophobic interactions with the photosystem. The thermodynamic analysis of the reactivity of K33E mutant towards PS I reveals that the effect of the mutation can be reversed by addition of magnesium cations, which probably bind at a place close to Glu33. The electrostatic surface potential does thus modulate the hydrophobic interactions with PS I by altering the solvent accessibility of some surface residues.
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PMID:Mutations in both leucine 12 and lysine 33 in plastocyanin from Synechocystis sp. PCC 6803 induce drastic changes in the hydrophobic interactions with Photosystem I. 1622 21

D-Alanine-D-alanine ligase (Ddl) is an important enzyme in the synthesis of bacterial peptidoglycan. The genes encoding Ddls from Escherichia coli K12 (EcDdlB), Oceanobacillus iheyensis JCM 11309 (OiDdl), Synechocystis sp. PCC 6803 (SsDdl) and Thermotoga maritima ATCC 43589 (TmDdl), the genomic DNA sequences of which have been determined, were cloned and the substrate specificities of these recombinant Ddls were investigated. Although OiDdl had a high substrate specificity for D-alanine; EcDdlB, SsDdl and TmDdl showed broad substrate specificities for D-serine, D-threonine, D-cysteine and glycine, in addition to D-alanine. Four D-amino acid dipeptides were produced using EcDdlB, and D-amino acid homo-dipeptides were successfully produced at high yields except for D-threonyl-D-threonine.
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PMID:D-Amino acid dipeptide production utilizing D-alanine-D-alanine ligases with novel substrate specificity. 1623 41

Degradation of the cyanobacterial light-harvesting antenna, the phycobilisome, is a general acclimation response that is observed under various stress conditions. In this study we identified a novel mutant of Synechococcus elongatus PCC 7942 that exhibits impaired phycobilisome degradation specifically during nitrogen starvation, unlike previously described mutants, which exhibit aberrant degradation under nitrogen, sulfur, and phosphorus starvation conditions. The phenotype of the new mutant, AldOmega, results from inactivation of ald (encoding alanine dehydrogenase). AldOmega is deficient in transcription induction of a number of genes during nitrogen starvation. These genes include the "general nutrient stress-related" genes, nblA and nblC, the products of which are essential for phycobilisome degradation. Furthermore, transcripts of several specific nitrogen-responsive genes accumulate at lower levels in AldOmega than in the wild-type strain. In contrast, ald inactivation did not decrease the accumulation of transcripts during sulfur starvation. Transcription of ald is induced upon nitrogen starvation, which is consistent with the ability of wild-type cells to maintain a low cellular content of alanine under these conditions. Unlike wild-type cells, AldOmega accumulates alanine upon nitrogen starvation. Our analyses suggest that alanine dehydrogenase activity is necessary for an adequate cellular response to nitrogen starvation. Decomposition of alanine may be required to provide a sufficient amount of ammonia. Furthermore, the accumulated alanine, or a related metabolite, may interfere with the cues that modulate acclimation during nitrogen starvation. Taken together, our results provide novel information regarding cellular responses to nitrogen starvation and suggest that mechanisms related to nitrogen-specific responses are involved in modulation of a general acclimation process.
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PMID:Alanine dehydrogenase activity is required for adequate progression of phycobilisome degradation during nitrogen starvation in Synechococcus elongatus PCC 7942. 1681 98

The cyanobacterium Synechocystis sp. PCC 6803 (S6803) expresses a two-on-two globin in which His46 (distal side) and His70 (proximal) function as heme iron axial ligands. His46 can be displaced by O2, CO, and CN-, among others, whereas His70 is not labile under native conditions. The residue preceding the proximal histidine has been implicated in controlling globin axial ligand reactivity; the details of the mechanism, however, are not well understood, and little information exists for bis-histidyl hexacoordinate proteins. In many vertebrate hemoglobins and in the Synechocystis protein, the position is occupied by an alanine, whereas, in myoglobins, it is a serine involved in an intricate hydrogen-bond network. We examined the role of Ala69 in S6803 hemoglobin through the effects of an Ala --> Ser replacement. The substitution resulted in minor structural perturbations, but the response of the holoprotein to temperature-, urea-, and acid-induced denaturation was measurably affected. Enhanced three-state behavior was manifested in the decoupling of heme binding and secondary-structure formation. Urea-gradient gel experiments revealed that the stability of the apoprotein was unchanged by the replacement and that a slight alteration of the folding kinetics occurred in the holoproteins. Cyanide-binding experiments were performed to assess trans effects. The apparent rate constant for association decreased 2-fold upon Ala69Ser replacement. This deceleration was attributed to a change in the lifetime of a state containing a decoordinated His46. The results demonstrated that, as in vertebrate globins and leghemoglobin, proximal influences operate to determine fundamental dynamic and thermodynamic properties of the protein.
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PMID:Proximal influences in two-on-two globins: effect of the Ala69Ser replacement on Synechocystis sp. PCC 6803 hemoglobin. 1698

Amino acid residue D1-Asp(170) of the D1-polypeptide of photosystem II was previously shown to be implicated in the binding and oxidation of the first manganese to be assembled into the Mn(4)Ca cluster of the oxygen-evolving complex (OEC). According to recent x-ray crystallographic structures of photosystem II, D1-Glu(333) is proposed to participate with D1-Asp(170) in the coordination of Mn4 of the OEC. Other residues in the C-terminal region of the D1-polypeptide are proposed to coordinate nearby manganese of the cluster. Site-directed replacements in Synechocystis sp. PCC 6803 at D1-His(332), D1-Glu(333), D1-Asp(342), D1-Ala(344), and D1-Ser(345) were examined with regard to their ability to influence the binding and oxidation of the first manganese in manganese-depleted photosystem II core complexes. Direct and indirect measurements reveal in all mutants, but most marked in D1-Glu(333) replaced by His, an impaired ability of Mn(2+) to reduce Y(Z)., indicating a reduced ability (elevated K(m)) compared with WT to bind and oxidize the first manganese of the OEC. The effect on the K(m) of these mutations is, however, considerably weaker than some of those constructed at D1-Asp(170) (replacement by Asn, Ala, and Ser). These observations imply that the C-terminal residues ultimately involved in manganese coordination contribute to the high affinity binding at D1-Asp(170) likely through electrostatic interactions. That these residues are far from D1-Asp(170) in the primary structure of the D1-polypeptide, imply that the C terminus of the D1-polypeptide is already close to its mature conformation at the first stages of assembly of the Mn(4)Ca cluster.
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PMID:Participation of the C-terminal region of the D1-polypeptide in the first steps in the assembly of the Mn4Ca cluster of photosystem II. 1720 88

In the recent X-ray crystallographic structural models of photosystem II, Asp342 of the D1 polypeptide is assigned as a ligand of the oxygen-evolving Mn4 cluster. To determine if D1-Asp342 ligates a Mn ion that undergoes oxidation during one or more of the S0 --> S1, S1 --> S2, and S2 --> S3 transitions, the FTIR difference spectra of the individual S state transitions in D1-D342N mutant PSII particles from the cyanobacterium Synechocystis sp. PCC 6803 were compared with those in wild-type PSII particles. Remarkably, the data show that the mid-frequency (1800-1200 cm-1) FTIR difference spectra of wild-type and D1-D342N PSII particles are essentially identical. Importantly, the mutation alters none of the carboxylate vibrational modes that are present in the wild-type spectra. The absence of significant mutation-induced spectral alterations in D1-D342N PSII particles shows that the oxidation of the Mn4 cluster does not alter the frequencies of the carboxylate stretching modes of D1-Asp342 during the S0 --> S1, S1 --> S2, or S2 --> S3 transitions. One explanation of these data is that D1-Asp342 ligates a Mn ion that does not increase its charge or oxidation state during any of these S state transitions. However, because the same conclusion was reached previously for D1-Asp170, and because the recent X-ray crystallographic structural models assign D1-Asp170 and D1-Asp342 as ligating different Mn ions, this explanation requires that (1) the extra positive charge that develops on the Mn4 cluster during the S1 --> S2 transition be localized on the Mn ion that is ligated by the alpha-COO- group of D1-Ala344 and (2) any increase in positive charge that develops on the Mn4 cluster during the S0 --> S1 and S2 --> S3 transitions be localized on the one Mn ion that is not ligated by D1-Asp170, D1-Asp342, or D1-Ala344. In separate experiments that were conducted with l-[1-13C]alanine, we found no evidence that D1-Asp342 ligates the same Mn ion that is ligated by the alpha-COO- group of D1-Ala344.
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PMID:No evidence from FTIR difference spectroscopy that aspartate-342 of the D1 polypeptide ligates a Mn ion that undergoes oxidation during the S0 to S1, S1 to S2, or S2 to S3 transitions in photosystem II. 1731 96

Anabaena sp. PCC 7120 is one of the few prokaryotes harboring a lipoxygenase (LOX) gene. The sequence resides in an open reading frame encoding a fusion protein of a catalase-like hemoprotein with an unusually short LOX (approximately 49 kDa) at the C terminus. The recombinant mini-LOX contains a non-heme iron in the active site and is highly active with linoleic and alpha-linolenic acids (which occur naturally in Anabaena) giving the respective 9R-hydroperoxides, the mirror image of the 9S-LOX products of plants. Using stereospecifically labeled [11-(3)H]linoleic acids we show that reaction is catalyzed via a typical antarafacial relationship of initial hydrogen abstraction and oxygenation. The mini-LOX oxygenated C16/C18:2-phosphatidylcholine with 9R specificity, suggesting a "tail first" mode of fatty acid binding. Site-directed mutagenesis of an active site Ala (Ala215), typically conserved as Gly in R-LOX, revealed that substitution with Gly retained 9R specificity, whereas the larger Val substitution switched oxygenation to 13S, implying that Ala215 represents the functional equivalent of the Gly in other R-LOX. Metabolism studies using a synthetic fatty acid with extended double bond conjugation, 9E,11Z,14Z-20:3omega6, showed that the mini-LOX can control oxygenation two positions further along the fatty acid carbon chain. We conclude that the mini-LOX, despite lacking the beta-barrel domain and much additional sequence, is catalytically complete. Interestingly, animal and plant LOX, which undoubtedly share a common ancestor, are related in sequence only in the catalytic domain; it is possible that the prokaryotic LOX represents a common link and that the beta-barrel domain was then acquired independently in the animal and plant kingdoms.
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PMID:A 49-kDa mini-lipoxygenase from Anabaena sp. PCC 7120 retains catalytically complete functionality. 1807 Aug 74

It is now quite well accepted that charge separation in PS2 reaction centers starts predominantly from the accessory chlorophyll B(A) and not from the special pair P(680). To identify spectral signatures of B(A,) and to further clarify the process of primary charge separation, we compared the femtosecond-infrared pump-probe spectra of the wild-type (WT) PS2 core complex from the cyanobacterium Synechocystis sp. PCC 6803 with those of two mutants in which the histidine residue axially coordinated to P(B) (D2-His(197)) has been changed to Ala or Gln. By analogy with the structure of purple bacterial reaction centers, the mutated histidine is proposed to be indirectly H-bonded to the C(9)=O carbonyl of the putative primary donor B(A) through a water molecule. The constructed mutations are thus expected to perturb the vibrational properties of B(A) by modifying the hydrogen bond strength, possibly by displacing the H-bonded water molecule, and to modify the electronic properties and the charge localization of the oxidized donor P(680)(+). Analysis of steady-state light-induced Fourier transform infrared difference spectra of the WT and the D2-His(197)Ala mutant indeed shows that a modification of the axially coordinating ligand to P(B) induces a charge redistribution of P(680)(+). In addition, a comparison of the time-resolved visible/midinfrared spectra of the WT and mutants has allowed us to investigate the changes in the kinetics of primary charge separation induced by the mutations and to propose a band assignment identifying the characteristic vibrations of B(A).
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PMID:Primary charge separation in the photosystem II core from Synechocystis: a comparison of femtosecond visible/midinfrared pump-probe spectra of wild-type and two P680 mutants. 1832 65

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