Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Synechococcus”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,045 records · Page 58Linked to original sources
PubMed1994

A small polypeptide triggers complete degradation of light-harvesting phycobiliproteins in nutrient-deprived cyanobacteria.

Phycobilisomes are the multiprotein complexes predominantly responsible for harvesting light energy in cyanobacteria and some eukaryotic algae. When the cyanobacterium Synechococcus sp. strain PCC 7942 is deprived of an essential nutrient, the phycobilisomes are specifically and rapidly degraded. Degradation may be either partial (after phosphorus deprivation) or complete (after sulfur or nitrogen deprivation). We have developed a visual screen to obtain mutants unable to degrade their phycobilisomes upon nutrient starvation. Complementation of one of these mutants led to the identification of a gene, designated nblA, that encodes a 59 amino acid polypeptide essential for phycobilisome degradation. Transcription of nblA increases dramatically in sulfur- or nitrogen-deprived cells and moderately in phosphorus-deprived cells. Using the phosphorus-regulated alkaline phosphatase (phoA) promoter as a tool, we engineered constructs from which we could control the expression of either sense or antisense nblA. Increased expression of sense nbLA caused complete phycobilisome degradation during phosphorus deprivation, while expression of antisense nblA prevented phycobilisome degradation. Hence, nblA is necessary, and may be sufficient, for the degradation of phycobilisomes under adverse environmental conditions. Further investigation of the mechanism by which nblA causes phycobilisome destruction may reveal general principles that govern the specificity of macromolecular complex degradation.

Alkaline Phosphatase↗
PubMed1983

Core substructure in cyanobacterial phycobilisomes.

The tricylindrical core of Synechocystis 6701 phycobilisomes is made up of four types of allophycocyanin-containing complexes: A, (alpha AP beta AP)3; B, (alpha AP beta AP)3 .10K; C, (alpha APB1 alpha AP2 beta AP3).10K; D, (alpha AP beta AP)2.18.5K.99K; where AP is allophycocyanin, APB is allophycocyanin B, and 10K, 18.5K, and 99K are polypeptides of 10,000, 18,500, and 99,000 daltons, respectively. The 18.5K polypeptide is a hitherto unrecognized biliprotein subunit with a single phycocyanobilin prosthetic group. The tricylindrical core is made up of 12 subcomplexes in the molar ratio of A:B:C:D: of 4:4:2:2. Complexes C and D act as terminal energy acceptors. From these results and previous analysis of the bicylindrical core of Synechococcus 6301 phycobilisomes [14,15] it is proposed that the two cylinders of the Synechocystis 6701 core, proximal to the thylakoid membrane, each have the composition ABCD, and that the distal cylinder has the composition A2B2.

Chromatography, DEAE-Cellulose↗
PubMed1996

The effect of sulfite on the ATP hydrolysis and synthesis activities in chloroplasts and cyanobacterial membrane vesicles can be explained by competition with phosphate.

The effect of sulfite on ATP synthesis and hydrolysis activities is investigated in spinach chloroplasts and in membrane vesicles from the cyanobacterium Synechococcus 6716. Sulfite inhibits phenazine methosulfate-mediated cyclic photophosphorylation both in thiol-modulated chloroplasts and in cyanobacterial membranes with HSO3- (bisulfite) as the active ionic species. The observed inhibition is not due to inhibition of electron transfer or to uncoupling by sulfite. ATP synthesis in cyanobacterial membranes is more sensitive to sulfite when the inorganic phosphate concentration is decreased. This indicates competition between sulfite and phosphate for the same binding site on the ATP synthase. In cyanobacterial membranes sulfite can replace a proton gradient as activator of ATP hydrolysis in the same way as in reduced chloroplasts. By modeling, competition between sulfite and phosphate can fully explain the findings concerning both inhibition and activation.

Adenosine Triphosphate↗
PubMed1998

Alteration of the alpha helix region of cyanobacterial ribulose 1,5-bisphosphate carboxylase/oxygenase to reflect sequences found in high substrate specificity enzymes.

The sequence at the alpha helix region of the eight-stranded beta/alpha barrel domain of the large subunit of Synechococcus sp. strain PCC 6301 ribulosebisphosphate carboxylase/oxygenase (rubisco) was altered by site-directed mutagenesis. Changes were made to match the corresponding residues in the rubisco large subunit of chromophytic and rhodophytic algae, which have considerably higher substrate specificity factors (ratio of the rate constants for the carboxylase and oxygenase reactions). A set of cumulative mutations of one to eight amino acid residues was prepared and examined and it was found that mutant enzymes which contained from one to five substitutions all exhibited substantial decreases in carboxylase activity. Mutant enzymes which contained from six to eight amino acid substitutions were inactive and failed to maintain their native quarternary structure. For enzymes which maintained their native structure, consecutive changes in the alpha helix 6 region yielded a progressive increase in the K(m) for ribulosebisphosphate, confirming the importance of this region in substrate binding. Despite these results, and previous studies which indicated the importance and potential of residues in the alpha helix 6 region to influence the ability of loop 6 to affect rubisco catalysis, simple cumulative substitution did not significantly alter the substrate specificity factor of the enzyme. The results of this study lend further credence to the idea that engineered enhancement of rubisco specificity will likely require coordination of alterations at multiple sites in the primary structure.

Amino Acid Sequence↗
PubMed1999

Extended heme promiscuity in the cyanobacterial cytochrome c oxidase: characterization of native complexes containing hemes A, O, and D, respectively.

The cyanobacteria Anacystis nidulans (Synechococcus sp. PCC6301), Synechocystis sp. PCC6803, Anabaena sp. PCC 7120, and Nostoc sp. PCC8009 were grown photoautotrophically under reduced oxygen tension in a medium with sulfate replaced by thiosulfate and nitrate replaced by ammonium as the S- and N-sources, respectively. In addition, Anabaena and Nostoc were grown under dinitrogen-fixing conditions in a medium free of combined nitrogen. Membranes were isolated from late-logarithmic cells (culture density corresponding to approximately 3 microliters packed cells per milliliter); cytoplasmic and thylakoid membranes were separated and purified according to established procedures. Acid-labile hemes were extracted from the membranes and subjected to reversed-phase high-performance liquid chromatography. Separated hemes were analyzed spectroscopically and identified by comparison with authentic standards. In addition to hemes B, A, and O, the latter of which was induced under semianaerobic conditions only, substitution of thiosulfate and ammonium for the oxy-anions sulfate and nitrate led to the appearance of spectrally discernible heme D in the membranes and extracts therefrom. However, spectroscopic and kinetic investigation of the membrane-bound heme D rather disproved any reaction with oxygen or carbon monoxide. Kinetic measurements performed with the membrane-bound respiratory oxidase gave evidence for only two kinetically competent terminal oxidases, a3 and o3, both apparently associated with a single type of apoprotein, viz. subunit I of the known cyanobacterial aa3-type cytochrome c oxidase. The heme D, on the other hand, seems to form a spectrally distinguished, yet kinetically ill-defined hemoprotein complex which does not qualify as a fully functional d-type terminal oxidase on our (wild-type) cyanobacteria even after growth under semianaerobic pseudo-reducing conditions. Also growth (of Anabaena and Nostoc) under dinitrogen-fixing conditions did not change this situation. Thus, we are left with (wild-type) cyanobacteria forming an unbranched respiratory chain with only a single type of terminal oxidase protein, viz. the known aa3-type cytochrome c oxidase. This oxidase, however, may incorporate different prosthetic (heme) groups in the sense of "heme promiscuity." Biosynthesis of the different heme groups thereby seems to respond to the ambient redox environment. In particular, however, conditions for expression of the two quinol oxidases potentially and additionally coded for by the genome of, e. g., Synechocystis sp. PCC6803 (see http://www.kazusa.or.jp/cyano), have not yet been found.

Acids↗
PubMed1998

Identification of biotinylation sites on proteins by selective retrieval of 2-iminobiotinylated peptides from proteolytic peptide mixtures: localization of the accessible lysine residues on the photosystem I subunits PsaD and PsaE.

An affinity purification technique was established that allows the selective isolation of 2-iminobiotinylated peptides from proteolytic digest of proteins in order to identify surface-exposed protein domains. Serving as model systems, two photosystem I subunits, PsaD and PsaE from the cyanobacterium Synechococcus elongatus, were overexpressed in Escherichia coli, modified in vitrowith NHS-2-iminobiotin which incorporates 2-iminobiotin at exposed amino groups, and subjected to proteolytic digestion by Glu-C and Arg-C protease, respectively. 2-Iminobiotin-containing proteolytic peptides were subsequently extracted from the proteolytic digests using avidin agarose in a batch procedure and the extracted peptides were separated by HPLC chromatography. The analysis of the peptide maps by electrospray ionization mass spectrometry or N-terminal sequencing showed that avidin-extracted peptide fractions contain almost exclusively 2-iminobiotinylated proteolytic fragments of PsaE or PsaD. No unmodified peptides of PsaD or PsaE were detected. According to this analysis, PsaE is accessible to biotinylation at all of its 7 lysine residues and at its N-terminus. Similarly, all 11 lysine residues of PsaD can be biotinylated and only the N-terminus of PsaD is not accessible.

Affinity Labels↗
PubMed1998

Measurement of photosystem I activity with photoreduction of recombinant flavodoxin.

Flavodoxin can function as an alternative electron acceptor for photosystem I (PSI) in place of ferredoxin under iron-limiting conditions. The isiB gene, encoding the flavodoxin in Synechococcus sp. PCC 7002, was overexpressed in Escherichia coli. Under the conditions employed, most recombinant flavodoxin (rFlvd) was in soluble form with cofactor correctly inserted. The absorption spectrum of rFlvd was identical to that of the native flavodoxin of the cyanobacteria. Photoreduction of rFlvd by PSI particles and thylakoid membranes was determined directly by monitoring the absorption change at 467 nm. The optimal conditions for rFlvd photoreduction were determined. Compared to other methods currently employed to measure PSI activity such as oxygen uptake in the presence of methyl viologen and NADP+ photoreduction in the presence of ferredoxin and ferredoxin:NADP+ oxidoreductase, measurement of PSI activity with flavodoxin as an electron acceptor has several advantages. It measures the full-chain electron transfer chain of PSI since flavodoxin accepts electrons from FA/FB and it is much simpler than the method with NADP+ photoreduction. With this method, we found that the affinity of wild-type PSI for rFlvd was 35% higher than that of the PsaE-less PSI, showing that this method is sensitive to structural changes of PSI. Our results demonstrate that rFlvd photoreduction is an effective and simple method for PSI activity measurement.

Bacterial Proteins↗
PubMed1994

Immunological and functional localization of both F-type and P-type ATPases in cyanobacterial plasma membranes.

Plasma and thylakoid membranes were prepared and purified from cyanobacteria Anacystis nidulans (Synechococcus PCC6301), Synechocystis PCC6803, and Anabaena PCC7120 grown photoautotrophically in axenic batch cultures and harvested either during early exponential growth (< 1 microliter packed cell mass/ml medium; light-saturated cells) or during linear growth (2-3 microliters packed cell mass/ml medium; light-limited cells). ATPase activities of methanol-activated membranes were determined by measuring (a) total inorganic phosphate released from added ATP, or (b) 32P(i) released from added [gamma-32P] ATP, or (c) NADH oxidation in a coupled ATPase/pyruvate kinase/lactate dehydrogenase system. In addition, membrane proteins were immunoblotted with antibodies raised against chloroplast and mitochondrial F1 coupling factor beta subunits, and Arabidopsis plasma membrane (P-type) ATPase, respectively. The results indicate the presence of (constitutive) P-type ATPase, inhibited by dicyclohexyl carbodiimide (DCCD), o-vanadate and diethylstilbestrol (DES) but insensitive to 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD), and of (inducible) F-type ATPase, inhibited by DCCD and NBD but insensitive to o-vanadate and DES, in the plasma membrane. Thylakoid membranes on the other hand contain F-type ATPase only. Our results emphasize the importance of plasma membrane energization even in obligately phototrophic cyanobacteria.

4-Chloro-7-nitrobenzofurazan↗
PubMed1994

The effect of sulfite on the ATP hydrolysis and synthesis activity of membrane-bound H(+)-ATP synthase from various species.

The action of sulfite on ATP hydrolysis and synthesis activities is investigated in membrane vesicles prepared from the cyanobacterium Synechococcus 6716, chromatophores from the photosynthetic purple bacterium Rhodospirillum rubrum, membrane vesicles from the related non-photosynthetic bacterium Paracoccus denitrificans, and bovine heart submitochondrial particles. Without any further pretreatment ATP hydrolysis is stimulated by sulfite in all four membrane preparations. Typically ATP synthesis in the cyanobacterial membrane vesicles is inhibited by sulfite, whereas ATP synthesis in chromatophores and the submitochondrial particles is not. These differences in sensitivity of ATP synthesis to sulfite, however, correspond well with the distribution of (photosynthetic) sulfur oxidizing pathways in the remaining three organisms/organelles compared in this study.

Adenosine Triphosphate↗
PubMed1999

Light-dependent and rhythmic psbA transcripts in homologous/heterologous cyanobacterial cells.

The psbA2 gene exhibits light-dependent and rhythmic expression in a unicellular cyanobacterium, Microcystis aeruginosa (Synechocystis) K-81. To further understand the psbA2 expression, biological analyses were performed in homologous and heterologous cyanobacterial cells. The results of the experiments using the K-81 cells revealed that (i) the light-dependent expression appeared on transcriptional and/or post-transcriptional level(s) under light/dark cycles, (ii) circadian-rhythmic transcripts were also observed under the control of an endogenous clock. To assess whether light-dependent and rhythmic psbA2 expression occurs in heterologous cyanobacterium, Synechococcus sp. strain PCC 7942, the K-81 psbA2 5'-upstream region of which the promoter and its around sequences share with those of PCC 7942 psbAII, was fused to the bacterial lacZ reporter gene, introduced into the genome of PCC 7942 and the psbA2 transcripts were directly investigated by primer extension. The K-81 psbA2 specific transcripts were also light-dependent and rhythmic in PCC 7942, strongly demonstrating that a common regulatory mechanism exists per se for the psbA2 expression in both strains. Furthermore, psbA2 expression in the recombinant PCC 7942 strain, AG400 in which the region from -404 to +111 of psbA2 is fused to lacZ, exhibited clear rhythmicity, while very little or no rhythmicity was observed in AG429 (-38 to +14, the only promoter region), suggesting that the region(s) around the promoter was essentially required for clear rhythmic expression.

Cyanobacteria↗
PubMed1995

Evolution of the phycobiliproteins.

Amino acid sequence alignments and phylogenetic analyses have been used to examine the relationships among 100 phycobiliprotein sequences. The alignments revealed a number of highly conserved amino acid residues that are involved in chromophore attachment and conformation, alpha-beta interactions and phycobilisome assembly. The phylogenetic analysis confirmed that the phycobiliprotein subfamilies, previously classified by their biochemical and spectroscopic properties, also formed coherent evolutionary groups. The alpha and beta subunits formed two distinct evolutionary lines that originate from a common ancestor. The pattern of divergence among the alpha subfamilies was identical to that of the beta subfamilies, strongly suggesting that the alpha and beta subunits of each phycobiliprotein type have coevolved. The phylogenetic data support a monophyletic separation of the eukaryotic sequences from the extant cyanobacterial sequences. The eukaryotic phycoerythrins appeared more closely related to the marine Synechococcus phycoerythrins than to the other cyanobacterial phycoerythrins. The cryptophyte phycobiliproteins formed a monophyletic group within the rhodophyte lineage. In conjunction with other phylogenetic markers, the analysis of additional phycobiliprotein sequences may help to further resolve the relationships among phycobiliprotein-containing organisms.

Amino Acid Sequence↗
PubMed1998

Crystal structure of human recombinant ornithine aminotransferase.

Ornithine aminotransferase (OAT), a pyridoxal-5'-phosphate dependent enzyme, catalyses the transfer of the delta-amino group of L-ornithine to 2-oxoglutarate, producing L-glutamate-gamma-semialdehyde, which spontaneously cyclizes to pyrroline-5-carboxylate, and L-glutamate. The crystal structure determination of human recombinant OAT is described in this paper. As a first step, the structure was determined at low resolution (6 A) by molecular replacement using the refined structure of dialkylglycine decarboxylase as a search model. Crystallographic phases were then refined and extended in a step-wise fashion to 2.5 A by cyclic averaging of the electron density corresponding to the three monomers within the asymmetric unit. Interpretation of the resulting map was straightforward and refinement of the model resulted in an R-factor of 17.1% (Rfree=24.3%). The success of the procedure demonstrates the power of real-space molecular averaging even with only threefold redundancy. The alpha6-hexameric molecule is a trimer of intimate dimers with a monomer-monomer interface of 5500 A2 per subunit. The three dimers are related by an approximate 3-fold screw axis with a translational component of 18 A. The monomer fold is that of a typical representative of subgroup 2 aminotransferases and very similar to those described for dialkylglycine decarboxylase from Pseudomonas cepacia and glutamate-1-semialdehyde aminomutase from Synechococcus. It consists of a large domain that contributes most to the subunit interface, a C-terminal small domain most distant to the 2-fold axis and an N-terminal region that contains a helix, a loop and a three stranded beta-meander embracing a protrusion in the large domain of the second subunit of the dimer. The large domain contains the characteristic central seven-stranded beta-sheet (agfedbc) covered by eight helices in a typical alpha/beta fold. The cofactor pyridoxal-5'-phosphate is bound through a Schiff base to Lys292, located in the loop between strands f and g. The C-terminal domain includes a four-stranded antiparallel beta-sheet in contact with the large domain and three further helices at the far end of the subunit. The active sites of the dimer lie, about 25 A apart, at the subunit and domain interfaces. The conical entrances are on opposite sides of the dimer. In the active site, R180, E235 and R413 are probable substrate binding residues. Structure-based sequence comparisons with related transaminases in this work support that view. In patients suffering from gyrate atrophy, a recessive hereditary genetic disorder that can cause blindness in humans, ornithine aminotransferase activity is lacking. A large number of frameshift and point mutations in the ornithine aminotransferase gene have been identified in such patients. Possible effects of the various point mutations on the structural stability or the catalytic competence of the enzyme are discussed in light of the three-dimensional structure.

Binding Sites↗
PubMed1998

Surface analysis of the photosystem I complex by electron and atomic force microscopy.

Two-dimensional (2D) crystals of the photosystem I (PSI) reaction center from Synechococcus sp. OD24 were analyzed by electron and atomic force microscopy. Surface relief reconstructions from electron micrographs of freeze-dried unidirectionally shadowed samples and topographs recorded with the atomic force microscope (AFM) provided a precise definition of the lumenal and stromal PSI surfaces. The lumenal surface was composed of four protrusions that surrounded an indentation. One of the protrusions, the PsaF subunit, was often missing. Removal of the extrinsic proteins with the AFM stylus exposed the stromal side of the PSI core, whose surface structure could then be imaged at a resolution better than 1.4 nm. This interfacial surface between core and extrinsic subunits, had a pseudo-2-fold symmetry and protrusions that correlated with the surface helices e and e' or were at the sites of putative alpha-helix-connecting loops estimated from the 4 A map of the complex. The molecular dissection achieved with the AFM, opens new possibilities to unveil the interfaces between subunits of supramolecular assemblies.

Amino Acid Sequence↗
PubMed1999

Refined structures of oxidized flavodoxin from Anacystis nidulans.

Flavodoxin from Anacystis nidulans (Synechococcus PCC 7942) was the first member of the flavodoxin family to be characterized, and is the structural prototype for the "long-chain" flavodoxins that have molecular masses of approximately 20 kDa. Crystal structure analyses and refinements of three orthorhombic forms of oxidized A. nidulans flavodoxin are reported, and salient features of the fold and the FMN binding site are compared with other flavodoxins. The structure of form I (wild-type: P212121, a=57.08 A, b=69.24 A, c=45.55 A), determined initially by multiple isomorphous replacement, has been refined to R=0.183 and R(free)=0.211 for data from 10.0 to 1.7 A resolution. Structures of form II (wild-type: P212121, a=60.05 A, b=65.85 A, c=51.36 A) and form III (Asn58Gly: P212121, a=51.30 A, b=59.15 A, c=94.44 A) have been determined by molecular replacement and refined versus data to 2.0 A and 1.85 A, respectively; the R values for forms II and III are 0.147 and 0.150. Changes in the molecular contacts that produce the alternative packings in these crystalline forms are analyzed. Deletion of the Asn side-chain in the mutant Asn58Gly removes an intermolecular stacking interaction and allows the alternative packing found in form III crystals. The functionally important 50's loop of the FMN binding site is less restrained by intermolecular contacts in these crystals but maintains the same conformation as in oxidized wild type protein. The structures reported here provide the starting point for structure-function studies of the reduced states and of mutants, described in the accompanying paper.

Amino Acid Sequence↗
PubMed1999

Comparisons of wild-type and mutant flavodoxins from Anacystis nidulans. Structural determinants of the redox potentials.

The long-chain flavodoxins, with 169-176 residues, display oxidation-reduction potentials at pH 7 that vary from -50 to -260 mV for the oxidized/semiquinone (ox/sq) equilibrium and are -400 mV or lower for the semiquinone/hydroquinone (sq/hq) equilibrium. To examine the effects of protein interactions and conformation changes on FMN potentials in the long-chain flavodoxin from Anacystis nidulans (Synechococcus PCC 7942), we have determined crystal structures for the semiquinone and hydroquinone forms of the wild-type protein and for the mutant Asn58Gly, and have measured redox potentials and FMN association constants. A peptide near the flavin ring, Asn58-Val59, reorients when the FMN is reduced to the semiquinone form and adopts a conformation ("O-up") in which O 58 hydrogen bonds to the flavin N(5)H; this rearrangement is analogous to changes observed in the flavodoxins from Clostridium beijerinckii and Desulfovibrio vulgaris. On further reduction to the hydroquinone state, the Asn58-Val59 peptide in crystalline wild-type A. nidulans flavodoxin rotates away from the flavin to the "O-down" position characteristic of the oxidized structure. This reversion to the conformation found in the oxidized state is unusual and has not been observed in other flavodoxins. The Asn58Gly mutation, at the site which undergoes conformation changes when FMN is reduced, was expected to stabilize the O-up conformation found in the semiquinone oxidation state. This mutation raises the ox/sq potential by 46 mV to -175 mV and lowers the sq/hq potential by 26 mV to -468 mV. In the hydroquinone form of the Asn58Gly mutant the C-O 58 remains up and hydrogen bonded to N(5)H, as in the fully reduced flavodoxins from C. beijerinckii and D. vulgaris. The redox and structural properties of A. nidulans flavodoxin and the Asn58Gly mutant confirm the importance of interactions made by N(5) or N(5)H in determining potentials, and are consistent with earlier conclusions that conformational energies contribute to the observed potentials.The mutations Asp90Asn and Asp100Asn were designed to probe the effects of electrostatic interactions on the potentials of protein-bound flavin. Replacement of acidic by neutral residues at positions 90 and 100 does not perturb the structure, but has a substantial effect on the sq/hq equilibrium. This potential is increased by 25-41 mV, showing that electrostatic interaction between acidic residues and the flavin decreases the potential for conversion of the neutral semiquinone to the anionic hydroquinone. The potentials and the effects of mutations in A. nidulans flavodoxin are rationalized using a thermodynamic scheme developed for C. beijerinckii flavodoxin.

Amino Acid Sequence↗
PubMed2001

Use of octyl beta-thioglucopyranoside in two-dimensional crystallization of membrane proteins.

A great interest exists in producing and/or improving two-dimensional (2D) crystals of membrane proteins amenable to structural analysis by electron crystallography. Here we report on the use of the detergent n-octyl beta-d-thioglucopyranoside in 2D crystallization trials of membrane proteins with radically different structures including FhuA from the outer membrane of Escherichia coli, light-harvesting complex II from Rubrivivax gelatinosus, and Photosystem I from cyanobacterium Synechococcus sp. We have analyzed by electron microscopy the structures reconstituted after detergent removal from lipid-detergent or lipid-protein-detergent micellar solutions containing either only n-octyl beta-d-thioglucopyranoside or n-octyl beta-d-thioglucopyranoside in combination with other detergents commonly used in membrane protein biochemistry. This allowed the definition of experimental conditions in which the use of n-octyl beta-d-thioglucopyranoside could induce a considerable increase in the size of reconstituted membrane structures, up to several micrometers. An other important feature was that, in addition to reconstitution of membrane proteins into large bilayered structures, this thioglycosylated detergent also was revealed to be efficient in crystallization trials, allowing the proteins to be analyzed in large coherent two-dimensional arrays. Thus, inclusion of n-octyl beta-d-thioglucopyranoside in 2D crystallization trials appears to be a promising method for the production of large and coherent 2D crystals that will be valuable for structural analysis by electron crystallography and atomic force microscopy.

Bacterial Outer Membrane Proteins↗
PubMed1997

Phytoene desaturase: heterologous expression in an active state, purification, and biochemical properties.

Conditions were developed for heterologous expression in Escherichia coli of the membrane-bound cyanobacterial/plant-type phytoene desaturase (PDS) from Synechococcus in an active form. Decrease of growth temperature for the transformant to 28 degrees C resulted in an increase of proteins in a supernatant fraction obtained after pressure disruption (20 MPa) of cells and centrifugation. This supernatant in which the highest PDS activity was found was used for purification to a homogeneous protein by ammonium sulfate precipitation and DEAE chromatography. The purified PDS was employed to determine substrate specificity and cofactor requirement. Substrates in addition to phytoene were phytofluene and 1,2-epoxy phytoene which were converted to zeta-carotene and the corresponding 1,2-epoxide. The reaction was stimulated by NAD, NADP, and oxygen. The K(m) values determined for phytoene and NADP were 3.5 microM and 14.3 mM, respectively.

Carotenoids↗
PubMed1993

Isolation of a delta 6-desaturase gene from the cyanobacterium Synechocystis sp. strain PCC 6803 by gain-of-function expression in Anabaena sp. strain PCC 7120.

The enzyme delta 6-desaturase is responsible for the conversion of linoleic acid (18:2) to gamma-linolenic acid (18:3 gamma). A cyanobacterial gene encoding delta 6-desaturase was cloned by expression of a Synechocystis genomic cosmid library in Anabaena, a cyanobacterium lacking delta 6-desaturase. Expression of the Synechocystis delta 6-desaturase gene in Anabaena resulted in the accumulation of gamma-linolenic acid (GLA) and octadecatetraenoic acid (18:4). The predicted 359 amino acid sequence of the Synechocystis delta 6-desaturase shares limited, but significant, sequence similarity with two other reported desaturases. Analysis of three overlapping cosmids revealed a delta 12-desaturase gene linked to the delta 6-desaturase gene. Expression of Synechocystis delta 6- and delta 12-desaturases in Synechococcus, a cyanobacterium deficient in both desaturases, resulted in the production of linoleic acid and gamma-linolenic acid.

Amino Acid Sequence↗