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PubMed1994

A carbonic anhydrase from the archaeon Methanosarcina thermophila.

Carbonic anhydrase (CA) from acetate-grown Methanosarcina thermophila was purified > 10,000-fold (22% recovery) to apparent homogeneity with a specific activity of 4872 units/mg. The estimated native molecular mass of the enzyme is 84 kDa based on gel filtration chromatography. SDS/PAGE revealed one protein band with an apparent molecular mass of 40 kDa. The M. thermophila CA is less sensitive than human CA isozyme II toward inhibition by sulfonamides and monovalent ions. The gene encoding this CA was cloned into pUC18 and sequenced. Escherichia coli harboring the recombinant plasmid expresses CA activity (2.3 units/mg of cell extract protein). Comparison of the deduced amino acid sequence with the N-terminal sequence of the purified protein shows that the gene encodes an additional 34 N-terminal residues with properties characteristic of signal peptides in secretory proteins. The calculated molecular mass (22.9 kDa) and pI (4.0) suggest that SDS/PAGE overestimates the subunit size and that the native enzyme is a tetramer. To our knowledge, the deduced amino acid sequence has no significant identity to any known CA but has 35% sequence identity to the first 197 deduced N-terminal amino acids of a proposed CO2-concentrating-mechanism protein from Synechococcus PCC7942 and 28% sequence identity to the deduced sequence of ferripyochelin binding protein from Pseudomonas aeruginosa. Thus, our results indicate that this archaeal CA represents a distinct class of CAs and provide a basis to determine physiological roles for CA in acetotrophic anaerobes.

Amino Acid Sequence↗
PubMed1994

High-level expression of human superoxide dismutase in the cyanobacterium Anacystis nidulans 6301.

A chemically synthesized gene encoding human CuZn superoxide dismutase (hSOD) was cloned into the shuttle vector pBAX18R and expressed in Anacystis nidulans 6301 (Synechococcus sp. strain PCC 6301) under the control of a ribulose-1,5-bisphosphate carboxylase/oxygenase gene (rbc) promoter derived from A. nidulans 6301. The sequences immediately upstream from the hSOD coding region and the distances between the ribosomal binding site and ATG initiation codon strongly affected the expression of the hSOD gene in A. nidulans cells. Optimal expression of hSOD was obtained with the expression vector pBAXSOD8-I, which contained a GGAGAG sequence. In defined conditions, irradiation with light increased hSOD enzyme activity in the transformants > 18-fold and the level of the hSOD protein reached a value of about 3% of the total soluble protein. The transformants that expressed hSOD acquired the ability to extenuate photooxidative damage induced by methyl viologen.

Amino Acid Sequence↗
PubMed1995

Light-generated nuclear quantum beats: a signature of photosynthesis.

Light-induced radical pairs in deuterated and deuterated plus 15N-substituted Synechococcus lividus cyanobacteria have been studied by transient EPR following pulsed laser excitation. Nuclear quantum beats are observed in the transverse electron magnetization at lower temperatures. Model calculations for the time profiles, evaluated at the high-field emissive maximum of the spectrum, indicate assignment of these coherences to nitrogen nuclei in the primary donor. Thorough investigation of the nuclear modulation patterns can provide detailed information on the electronic structure of the primary donor, providing insight into the mechanism of the primary events of plant photosynthesis.

Journal Article↗
PubMed1995

Cyanobacterial protein with similarity to the chlorophyll a/b binding proteins of higher plants: evolution and regulation.

We have isolated, from the prokaryotic cyanobacterium Synechococcus sp. strain PCC 7942, a gene encoding a protein of 72 amino acids [designated high light inducible protein (HLIP)] with similarity to the extended family of eukaryotic chlorophyll a/b binding proteins (CABs). HLIP has a single membrane-spanning alpha-helix, whereas both the CABs and the related early light inducible proteins have three membrane-spanning helices. Hence, HLIP may represent an evolutionary progenitor of the eukaryotic members of the CAB extended family. We also show that the gene encoding HLIP is induced by high light and blue/UV-A radiation. The evolution, regulation, and potential function of HLIP are discussed.

Amino Acid Sequence↗
PubMed1997

Crystal structure of glutamate-1-semialdehyde aminomutase: an alpha2-dimeric vitamin B6-dependent enzyme with asymmetry in structure and active site reactivity.

The three-dimensional structure of glutamate-1-semialdehyde aminomutase (EC 5.4.3.8), an alpha2-dimeric enzyme from Synechococcus, has been determined by x-ray crystallography using heavy atom derivative phasing. The structure, refined at 2.4-A resolution to an R-factor of 18.7% and good stereochemistry, explains many of the enzyme's unusual specificity and functional properties. The overall fold is that of aspartate aminotransferase and related B6 enzymes, but it also has specific features. The structure of the complex with gabaculine, a substrate analogue, shows unexpectedly that the substrate binding site involves residues from the N-terminal domain of the molecule, notably Arg-32. Glu-406 is suitably positioned to repel alpha-carboxylic acids, thereby suggesting a basis for the enzyme's reaction specificity. The subunits show asymmetry in cofactor binding and in the mobilities of the residues 153-181. In the unliganded enzyme, one subunit has the cofactor bound as an aldimine of pyridoxal phosphate with Lys-273 and, in this subunit, residues 153-181 are disordered. In the other subunit in which the cofactor is not covalently bound, residues 153-181 are well defined. Consistent with the crystallographically demonstrated asymmetry, a form of the enzyme in which both subunits have pyridoxal phosphate bound to Lys-273 through a Schiff base showed biphasic reduction by borohydride in solution. Analysis of absorption spectra during reduction provided evidence of communication between the subunits. The crystal structure of the reduced form of the enzyme shows that, despite identical cofactor binding in each monomer, the structural asymmetry at residues 153-181 remains.

Amino Acid Sequence↗
PubMed1998

An SmtB-like repressor from Synechocystis PCC 6803 regulates a zinc exporter.

ORF slr0798, now designated ziaA, from Synechocystis PCC 6803 encodes a polypeptide with sequence features of heavy metal transporting P-type ATPases. Increased Zn2+ tolerance and reduced 65Zn accumulation was observed in Synechococcus PCC 7942, strain R2-PIM8(smt), containing ziaA and upstream regulatory sequences, compared with control cells. Conversely, reduced Zn2+ tolerance was observed following disruption of ziaA in Synechocystis PCC 6803, and ziaA-mediated restoration of Zn2+ tolerance has subsequently been used as a selectable marker for transformation. Nucleotide sequences upstream of ziaA, fused to a promoterless lacZ gene, conferred Zn2+-dependent beta-galactosidase activity when introduced into R2-PIM8(smt). The product of ORF sll0792, designated ZiaR, is a Zn2+-responsive repressor of ziaA transcription. Reporter gene constructs lacking ziaR conferred elevated Zn2+-independent expression from the ziaA operator-promoter in R2-PIM8(smt). Gel retardation assays detected ZiaR-dependent complexes forming with the zia operator-promoter and ZiaR-DNA binding was enhanced by treatment with a metal-chelator in vitro. Two mutants of ZiaR (C71S/C73S and H116R) bound to, and repressed expression from, the ziaA operator-promoter but were unable to sense Zn2+. Metal coordination to His-imidazole and Cys-thiolate ligands at these residues of ZiaR is thus implicated in Zn2+-perception by Synechocystis PCC 6803.

Bacterial Proteins↗
PubMed2000

Nucleotide binding and autophosphorylation of the clock protein KaiC as a circadian timing process of cyanobacteria.

A negative feedback control of kaiC expression by KaiC protein has been proposed to generate a basic oscillation of the circadian clock in the cyanobacterium Synechococcus sp. PCC 7942. KaiC has two P loops or Walker's motif As, that are potential ATP-/GTP-binding motifs and DXXG motifs conserved in various GTP-binding proteins. Herein, we demonstrate that in vitro KaiC binds ATP and, with lower affinity, GTP. Point mutation by site-directed mutagenesis of P loop 1 completely nullified the circadian rhythm of kaiBC expression and markedly reduced ATP-binding activity. Moreover, KaiC can be autophosphorylated in vitro. These results suggest that the nucleotide-binding activity of KaiC plays important roles in the generation of circadian oscillation in cyanobacteria.

Adenosine Triphosphate↗
PubMed1995

A SecY homolog in Arabidopsis thaliana. Sequence of a full-length cDNA clone and import of the precursor protein into chloroplasts.

Proteins are translocated across the thylakoid membrane by two distinct pathways in higher plant chloroplasts, one of which is related to prokaryotic Sec-dependent translocation mechanisms. SecY is an essential, hydrophobic component of the membrane-bound translocase complex in bacteria, and we report here the nucleotide sequence of a full-length cDNA encoding a homolog of SecY from Arabidopsis thaliana. The predicted protein of 551 residues includes an amino-terminal extension of approximately 120 residues when compared with other SecY proteins. The deduced sequence of the mature protein, cpSecY, is 41% identical with SecY from Synechococcus and 33% identical with the Escherichia coli protein. The extension serves to target the protein into chloroplasts; transcription-translation of the cDNA yields a 58-kDa precursor protein which is imported into pea chloroplasts, processed to a product of 46 kDa, and targeted into the thylakoid membrane.

Amino Acid Sequence↗
PubMed1995

Isolation and characterization of a cDNA encoding the SecA protein from spinach chloroplasts. Evidence for azide resistance of Sec-dependent protein translocation across thylakoid membranes in spinach.

Thylakoid membranes of chloroplasts in higher plants harbor different pathways for the translocation of proteins. One of these routes is related to the prokaryotic Sec pathway, which mediates the secretion of particular proteins into the periplasmic space and involves the SecA protein as an essential component. We have isolated a full size cDNA of 3739 nucleotides encoding the SecA homologue from spinach. It contains an open reading frame of 1036 codons corresponding to a polypeptide with a calculated mass of 117 kDa. The deduced amino acid sequence shows between 43 and 49% identity to SecA proteins from bacteria and lower algae and 62% identity to SecA of the cyanobacterium Synechococcus sp. PCC7942. Compared with the Escherichia coli protein, spinach SecA carries an amino-terminal extension of approximately 80 residues. In organello experiments performed with the protein made in vitro by transcription of the cDNA and cell-free translation of the resulting RNA showed that this extension comprises a transit peptide that mediates the import of the protein into the chloroplast. The processed product of approximately 107 kDa accumulates predominantly in the stroma and to a lower extent associates with the thylakoid membrane. Comparably to E. coli, in which SecA activity can be inhibited by sodium azide, thylakoid translocation of a subset of lumenal proteins is sensitive to sodium azide in pea but not in spinach chloroplasts, suggesting that the latter contain an azide-resistant SecA variant.

Adenosine Triphosphatases↗
PubMed1995

Transient accumulation of heme O (cytochrome o) in the cytoplasmic membrane of semi-anaerobic Anacystis nidulans. Evidence for oxygenase-catalyzed heme O/A transformation.

Incubation of obligately photoautotrophic and aerobic cyanobacterium Anacystis nidulans (Synechococcus sp. PCC 6301) in the light in the presence of the photo-system II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea and equilibrated with approximately 1% (v/v) O2 in N2 (10 microM O2 in solution) led to a decrease of the heme A content of isolated cytoplasmic membranes and to the appearance of heme O. The latter was not seen in membranes from fully aerated cells (> 210 microM dissolved O2). Non-covalently bound hemes extracted from the membranes were identified by reversed phase high performance liquid chromatography. Heme A and O contents of the membranes changed in a reversible fashion solely depending on the ambient oxygen regime. Both hemes A and O combine with the same apoprotein as suggested by immunoblotting. CO/reduced-minus-reduced optical difference spectra, photoaction spectra of CO-inhibited O2 uptake by the membranes, and pyridine hemochrome spectra pointed to either heme belonging to a functional form of the terminal oxidase. The NADH:O2 oxidoreductase reaction catalyzed by membranes from both high O2 and low O2 cells was strictly dependent on the addition of catalytic amounts of cytochrome c, fully inhibited by 1.2 microM KCN, and insensitive to 5 microM 2-n-heptyl-4-hydroxyquinoline-N-oxide. O2 uptake by the membranes was effectively catalyzed by N,N,N',N'-tetramethyl-p-phenylenediamine but not 2-methylnaphthoquinol or plastoquinol-1 as artificial substrates. Therefore we conclude that the cyanobacterial respiratory oxidase, irrespective of the type of heme in its O2-reducing center, is a cytochrome c rather than a quinol oxidase.

Anaerobiosis↗
PubMed1996

Comparison of primary charge separation in the photosystem II reaction center complex isolated from wild-type and D1-130 mutants of the cyanobacterium Synechocystis PCC 6803.

We compare primary charge separation in a photosystem II reaction center preparation isolated from a wild-type (WT) control strain of the cyanobacterium Synechocystis sp. PCC 6803 and from two site-directed mutants of Synechocystis in which residue 130 of the D1 polypeptide has been changed from a glutamine to either a glutamate (mutant D1-Gln130Glu), as in higher plant sequences, or a leucine residue (mutant D1-Gln130Leu). The D1-130 residue is thought to be close to the pheophytin electron acceptor. We show that, when P680 is photoselectively excited, the primary radical pair state P680+Ph- is formed with a time constant of 20-30 ps in the WT and both mutants; this time constant is very similar to that observed in Pisum sativum (a higher plant). We also show that a change in the residue at position D1-130 causes a shift in the peak of the pheophytin Qx-band. Nanosecond and picosecond transient absorption measurements indicate that the quantum yield of radical pair formation (phi RP), associated with the 20-30-ps component, is affected by the identify of the D1-130 residue. We find that, for the isolated photosystem II reaction center particle, phi RP higher plant > phi RP D1-Gln130Glu mutant > phi RP WT > phi RP D1-Gln130Leu mutant. Furthermore, the spectroscopic and quantum yield differences we observe between the WT Synechocystis and higher plant photosystem II, seem to be reversed by mutating the D1-130 ligand so that it is the same as in higher plants. This result is consistent with the previously observed natural regulation of quantum yield in Synechococcus PS II by particular changes in the D1 polypeptide amino acid sequence (Clark, A.K., Hurry, V. M., Gustafsson, P. and Oquist, G. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 11985-11989).

Cyanobacteria↗
PubMed1999

Photosystem I, an improved model of the stromal subunits PsaC, PsaD, and PsaE.

An improved electron density map of photosystem I (PSI) calculated at 4-A resolution yields a more detailed structural model of the stromal subunits PsaC, PsaD, and PsaE than previously reported. The NMR structure of the subunit PsaE of PSI from Synechococcus sp. PCC7002 (Falzone, C. J., Kao, Y.-H., Zhao, J., Bryant, D. A., and Lecomte, J. T. J. (1994) Biochemistry 33, 6052-6062) has been used as a model to interpret the region of the electron density map corresponding to this subunit. The spatial orientation with respect to other subunits is described as well as the possible interactions between the stromal subunits. A first model of PsaD consisting of a four-stranded beta-sheet and an alpha-helix is suggested, indicating that this subunit partly shields PsaC from the stromal side. In addition to the improvements on the stromal subunits, the structural model of the membrane-integral region of PSI is also extended. The current electron density map allows the identification of the N and C termini of the subunits PsaA and PsaB. The 11-transmembrane alpha-helices of these subunits can now be assigned uniquely to the hydrophobic segments identified by hydrophobicity analyses.

Amino Acid Sequence↗
PubMed1999

Localization of two phylloquinones, QK and QK', in an improved electron density map of photosystem I at 4-A resolution.

An improved electron density map of photosystem I from Synechococcus elongatus calculated at 4-A resolution for the first time reveals a second phylloquinone molecule and thereby completes the set of cofactors constituting the electron transfer system of this iron-sulfur type photosynthetic reaction center: six chlorophyll a, two phylloquinones, and three Fe4S4 clusters. The location of the newly identified phylloquinone pair, the individual plane orientations of these molecules, and the resulting distances to other cofactors of the electron transfer system are discussed and compared with those determined by magnetic resonance techniques.

Cyanobacteria↗
PubMed1999

Stoichiometry of proton release from the catalytic center in photosynthetic water oxidation. Reexamination by a glass electrode study at ph 5.5-7.2.

The catalytic center (CC) of water oxidation in photosystem II passes through four stepwise increased oxidized states (S(0)-S(4)) before O(2) evolution takes place from 2H(2)O in the S(4) --> S(0) transition. The pattern of the release of the four protons from the CC cannot be followed directly in the medium, because proton release from unknown amino acid residues also takes place. However, pH-independent net charge oscillations of 0:0:1:1 in S(0):S(1):S(2):S(3) have been considered as an intrinsic indicator for the H(+) release from the CC. The net charges have been proposed to be created as the charge difference between electron abstraction and H(+) release from the CC. Then the H(+) release from the CC is 1:0:1:2 for the S(0) --> S(1) --> S(2) --> S(3) --> S(0) transition. Strong support for this conclusion is given in this work with the analysis of the pH-dependent pattern of H(+) release in the medium measured directly by a glass electrode between pH 5.5 and 7.2. Improved and crystallizable photosystem II core complexes from the cyanobacterium Synechococcus elongatus were used as material. The pattern can be explained by protons released from the CC with a stoichiometry of 1:0:1:2 and protons from an amino acid group (pK approximately 5.7) that is deprotonated and reprotonated through electrostatic interaction with the oscillating net charges 0:0:1:1 in S(0):S(1):S(2):S(3). Possible water derivatives that circulate through the S states have been named.

Catalytic Domain↗
PubMed1999

Characterization of the gene encoding serine acetyltransferase, a regulated enzyme of cysteine biosynthesis from the protist parasites Entamoeba histolytica and Entamoeba dispar. Regulation and possible function of the cysteine biosynthetic pathway in Entamoeba.

The enteric protist parasites Entamoeba histolytica and Entamoeba dispar possess a cysteine biosynthetic pathway, unlike their mammalian host, and are capable of de novo production of L-cysteine. We cloned and characterized cDNAs that encode the regulated enzyme serine acetyltransferase (SAT) in this pathway from these amoebae by genetic complementation of a cysteine-auxotrophic Escherichia coli strain with the amoebic cDNA libraries. The deduced amino acid sequences of the amoebic SATs exhibited, within the most conserved region, 36-52% identities with the bacterial and plant SATs. The amoebic SATs contain a unique insertion of eight amino acids, also found in the corresponding region of a plasmid-encoded SAT from Synechococcus sp., which showed the highest overall identities to the amoebic SATs. Phylogenetic reconstruction also revealed a close kinship of the amoebic SATs with cyanobacterial SATs. Biochemical characterization of the recombinant E. histolytica SAT revealed several enzymatic features that distinguished the amoebic enzyme from the bacterial and plant enzymes: 1) inhibition by L-cysteine in a competitive manner with L-serine; 2) inhibition by L-cystine; and 3) no association with cysteine synthase. Genetically engineered amoeba strains that overproduced cysteine synthase and SAT were created. The cysteine synthase-overproducing amoebae had a higher level of cysteine synthase activity and total thiol content and revealed increased resistance to hydrogen peroxide. These results indicate that the cysteine biosynthetic pathway plays an important role in antioxidative defense of these enteric parasites.

Acetyltransferases↗
PubMed2000

Towards structural determination of the water-splitting enzyme. Purification, crystallization, and preliminary crystallographic studies of photosystem II from a thermophilic cyanobacterium.

A photosystem II preparation from the thermophilic cyanobacterium Synechococcus elongatus, which is especially suitable for three-dimensional crystallization in a fully active form was developed. The efficient purification method applied here yielded 10 mg of protein of a homogenous dimeric complex of about 500 kDa within 2 days. Detailed characterization of the preparation demonstrated a fully active electron transport chain from the manganese cluster to plastoquinone in the Q(B) binding site. The oxygen-evolving activity, 5000-6000 micromol of O(2)/(h.mg of chlorophyll), was the highest so far reported and is maintained even at temperatures as high as 50 degrees C. The crystals obtained by the vapor diffusion method diffracted to a resolution of 4.3 A. The space group was determined to be P2(1)2(1)2(1) with four photosystem II dimers per unit cell. Analysis of the redissolved crystals revealed that activity, supramolecular organization, and subunit composition were maintained during crystallization.

Chromatography, High Pressure Liquid↗
PubMed2001

A novel heat shock protein plays an important role in thermal stress management in cyanobacteria.

A novel heat shock gene, orf7.5, which encodes a putative acidic polypeptide of 63 amino acids, was cloned from the cyanobacterium Synechococcus sp. PCC 7942. Northern blot analysis revealed the presence of 400- and 330-base orf7.5 mRNAs, which were barely detectable in the cells grown at 30 degrees C but increased transiently in response to heat shock at 40 or 45 degrees C. Primer extension analysis showed that the two mRNAs have different 5'-ends. Chloramphenicol enhanced the accumulation of the orf7.5 mRNA, whereas it inhibited the increase in the amount of the groESL mRNA. To reveal the role of the orf7.5 gene in thermal stress management, we constructed a stable mutant in which a gene conferring resistance to an antibiotic was inserted into the coding region of the orf7.5 gene. The interruption led to a marked inhibition of growth at 45 degrees C and a decrease in the basal and acquired thermo-tolerances at 50 degrees C in the transformants, indicating that the gene plays a role in thermal stress management. The orf7.5 mutant could be complemented with a return to the wild type phenotype by a DNA fragment containing orf7.5 but not by mutated orf7.5s, in which a nonsense mutation was generated by introducing a frameshift or a point mutation within the orf7.5-coding region. Thus, thermo-tolerance requires an appropriate translation product, not simply a transcript. Accumulation of the groESL transcript in the orf7.5 mutant was strongly reduced, suggesting that the orf7.5 gene product controls the expression of the groESL operon.

Amino Acid Sequence↗
PubMed2001

Substrate water exchange in photosystem II depends on the peripheral proteins.

The (18)O exchange rates for the substrate water bound in the S(3) state were determined in different photosystem II sample types using time-resolved mass spectrometry. The samples included thylakoid membranes, salt-washed Triton X-100-prepared membrane fragments, and purified core complexes from spinach and cyanobacteria. For each sample type, two kinetically distinct isotopic exchange rates could be resolved, indicating that the biphasic exchange behavior for the substrate water is inherent to the O(2)-evolving catalytic site in the S(3) state. However, the fast phase of exchange became somewhat slower (by a factor of approximately 2) in NaCl-washed membrane fragments and core complexes from spinach in which the 16- and 23-kDa extrinsic proteins have been removed, compared with the corresponding rate for the intact samples. For CaCl(2)-washed membrane fragments in which the 33-kDa manganese stabilizing protein (MSP) has also been removed, the fast phase of exchange slowed down even further (by a factor of approximately 3). Interestingly, the slow phase of exchange was little affected in the samples from spinach. For core complexes prepared from Synechocystis PCC 6803 and Synechococcus elongatus, the fast and slow exchange rates were variously affected. Nevertheless, within the experimental error, nearly the same exchange rates were measured for thylakoid samples made from wild type and an MSP-lacking mutant of Synechocystis PCC 6803. This result could indicate that the MSP has a slightly different function in eukaryotic organisms compared with prokaryotic organisms. In all samples, however, the differences in the exchange rates are relatively small. Such small differences are unlikely to arise from major changes in the metal-ligand structure at the catalytic site. Rather, the observed differences may reflect subtle long range effects in which the exchange reaction coordinates become slightly altered. We discuss the results in terms of solvent penetration into photosystem II and the regional dielectric around the catalytic site.

Catalysis↗