Cyanobacteria as Nanogold Factories II: Chemical Reactivity and anti-Myocardial Infraction Properties of Customized Gold Nanoparticles Biosynthesized by Cyanothece sp.

Cyanothece sp., a coccoid, unicellular, nitrogen-fixing and hydrogen-producing cyanobacterium, has been used in this study to biosynthesize customized gold nanoparticles under certain chemical conditions. The produced gold nanoparticles had a characteristic absorption band at 525-535 nm. Two types of gold nanoparticle, the purple and blue, were formed according to the chemical environment in which the cyanobacterium was grown. Dynamic light scattering was implemented to estimate the size of the purple and blue nanoparticles, which ranged from 80 ± 30 nm and 129 ± 40 nm in diameter, respectively. The highest scattering of laser light was recorded for the blue gold nanoparticles, which was possibly due to their larger size and higher concentration. The appearance of anodic and cathodic peaks in cyclic voltammetric scans of the blue gold nanoparticles reflected the oxidation into gold oxide, followed by the subsequent reduction into the nano metal state. The two produced forms of gold nanoparticles were used to treat isoproterenol-induced myocardial infarction in experimental rats. Both forms of nanoparticles ameliorated myocardial infarction injury, with a slight difference in their curative activity with the purple being more effective. Mechanisms that might explain the curative effect of these nanoparticles on the myocardial infarction were proposed. The morphological, physiological, and biochemical attributes of the Cyanothece sp. cyanobacterium were fundamental for the successful production of "tailored" nanoparticles, and complemented the chemical conditions for the differential biosynthesis process. The present research represents a novel approach to manipulate cyanobacterial cells towards the production of different-sized gold nanoparticles whose curative impacts vary accordingly. This is the first report on that type of manipulated gold nanoparticles biosynthesis which will hopefully open doors for further investigations and biotechnological applications.

Broad-Spectrum Anti-Adhesive Coating Based on an Extracellular Polymer from a Marine Cyanobacterium.

Medical device-associated infections are a major health threat, representing about half of all hospital-acquired infections. Current strategies to prevent this problem based on device coatings with antimicrobial compounds (antibiotics or antiseptics) have proven to be insufficient, often toxic, and even promoting bacterial resistance. Herein, we report the development of an infection-preventive coating (CyanoCoating) produced with an extracellular polymer released by the marine cyanobacterium Cyanothece sp. CCY 0110. CyanoCoating was prepared by spin-coating and its bacterial anti-adhesive efficiency was evaluated against relevant etiological agents (Staphylococcus aureus, S. epidermidis, Pseudomonas aeruginosa and Escherichia coli) and platelets, both in the presence or absence of human plasma proteins. CyanoCoating cytotoxicity was assessed using the L929 fibroblasts cell line. CyanoCoating exhibited a smooth topography, low thickness and high hydrophilic properties with mild negative charge. The non-cytotoxic CyanoCoating prevented adhesion of all the bacteria tested (≤80%) and platelets (<87%), without inducing platelet activation (even in the presence of plasma proteins). The significant reduction in protein adsorption (<77%) confirmed its anti-adhesive properties. The development of this anti-adhesive coating is an important step towards the establishment of a new technological platform capable of preventing medical device-associated infections, without inducing thrombus formation in blood-contacting applications.

Analysis of Protein Complexes in the Unicellular Cyanobacterium Cyanothece ATCC 51142.

The unicellular cyanobacterium Cyanothece ATCC 51142 is capable of oxygenic photosynthesis and biological N2 fixation (BNF), a process highly sensitive to oxygen. Previous work has focused on determining protein expression levels under different growth conditions. A major gap of our knowledge is an understanding on how these expressed proteins are assembled into complexes and organized into metabolic pathways, an area that has not been thoroughly investigated. Here, we combined size-exclusion chromatography (SEC) with label-free quantitative mass spectrometry (MS) and bioinformatics to characterize many protein complexes from Cyanothece 51142 cells grown under a 12 h light-dark cycle. We identified 1386 proteins in duplicate biological replicates, and 64% of those proteins were identified as putative complexes. Pairwise computational prediction of protein-protein interaction (PPI) identified 74 822 putative interactions, of which 2337 interactions were highly correlated with published protein coexpressions. Many sequential glycolytic and TCA cycle enzymes were identified as putative complexes. We also identified many membrane complexes that contain cytoplasmic domains. Subunits of NDH-1 complex eluted in a fraction with an approximate mass of ∼669 kDa, and subunits composition revealed coexistence of distinct forms of NDH-1 complex subunits responsible for respiration, electron flow, and CO2 uptake. The complex form of the phycocyanin beta subunit was nonphosphorylated, and the monomer form was phosphorylated at Ser20, suggesting phosphorylation-dependent deoligomerization of the phycocyanin beta subunit. This study provides an analytical platform for future studies to reveal how these complexes assemble and disassemble as a function of diurnal and circadian rhythms.

Oxidation of the Cyanobactin Precursor Peptide Is Independent of the Leader Peptide and Operates in a Defined Order.

The five-membered nitrogen plus heteroatom rings known as azolines or in their oxidized form as azoles are very common in natural products and drugs. The oxidation of thiazoline to thiazole in the cyanobactin class of natural products is one of the several important transformations that are known to alter the biological properties of the compound. The ordering of the various chemical reactions that occur during cyanobactin biosynthesis is not fully understood. The structure of the flavin-dependent enzyme responsible for the oxidation of multiple thiazolines reveals it contains an additional domain that in other enzymes recognizes linear peptides. We characterize the activity of the enzyme on two substrates: one with a peptide leader and one without. Kinetics and biophysics reveal that the leader on the substrate is not recognized by the enzyme. The enzyme is faster on either substrate than the macrocyclase or protease in vitro. The enzyme has a preferred order of oxidation of multiple thiazolines in the same linear peptide.

High myristic acid content in the cyanobacterium Cyanothece sp. PCC 8801 results from substrate specificity of lysophosphatidic acid acyltransferase.

Analysis of fatty acids from the cyanobacterium Cyanothece sp. PCC 8801 revealed that this species contained high levels of myristic acid (14:0) and linoleic acid in its glycerolipids, with minor contributions from palmitic acid (16:0), stearic acid, and oleic acid. The level of 14:0 relative to total fatty acids reached nearly 50%. This 14:0 fatty acid was esterified primarily to the sn-2 position of the glycerol moiety of glycerolipids. This characteristic is unique because, in most of the cyanobacterial strains, the sn-2 position is esterified exclusively with C16 fatty acids, generally 16:0. Transformation of Synechocystis sp. PCC 6803 with the PCC8801_1274 gene for lysophosphatidic acid acyltransferase (1-acyl-sn-glycerol-3-phosphate acyltransferase) from Cyanothece sp. PCC 8801 increased the level of 14:0 from 2% to 17% in total lipids and the increase in the 14:0 content was observed in all lipid classes. These findings suggest that the high content of 14:0 in Cyanothece sp. PCC 8801 might be a result of the high specificity of this acyltransferase toward the 14:0-acyl-carrier protein.

Biolubricant potential of exopolysaccharides from the cyanobacterium Cyanothece epiphytica.

Exopolysaccaharides (EPS) are carbohydrate polymers secreted by microbial cells, as a protective layer termed sheath or capsule. Their composition is variable. Optimisation of nutrient factors and the effect of some simple stresses on the ability of Cyanothece epiphytica to produce EPS were tested. Of the tested stresses, exposure to ozone for 50 s at 0.06 mg/L resulted in a relatively high EPS yield, without any damage to cell structure. EPS was characterised physicochemically. Chemically, it was found to be composed of pentoses arabinose and xylose; hexoses glucose, galactose and mannose; and the deoxyhexose fucose sugars which were sulphated and with different functional groups. EPS from C. epiphytica was found to be a good hydrophobic dispersant, an excellent emulsifier as well as a flocculant. Its potential as a biolubricant with characteristics better than the conventional lubricant 'grease' was revealed through analysis. This study gave the clue for developing a commercial technology to produce a less expensive and more environment-friendly natural lubricant from the cyanobacterium C. epiphytica for tribological applications.

Bound Substrate in the Structure of Cyanobacterial Branching Enzyme Supports a New Mechanistic Model.

Branching enzyme (BE) catalyzes the formation of α-1,6-glucosidic linkages in amylopectin and glycogen. The reaction products are variable, depending on the organism sources, and the mechanistic basis for these different outcomes is unclear. Although most cyanobacteria have only one BE isoform belonging to glycoside hydrolase family 13, Cyanothece sp. ATCC 51142 has three isoforms (BE1, BE2, and BE3) with distinct enzymatic properties, suggesting that investigations of these enzymes might provide unique insights into this system. Here, we report the crystal structure of ligand-free wild-type BE1 (residues 5-759 of 1-773) at 1.85 Å resolution. The enzyme consists of four domains, including domain N, carbohydrate-binding module family 48 (CBM48), domain A containing the catalytic site, and domain C. The central domain A displays a (ß/α)8-barrel fold, whereas the other domains adopt ß-sandwich folds. Domain N was found in a new location at the back of the protein, forming hydrogen bonds and hydrophobic interactions with CBM48 and domain A. Site-directed mutational analysis identified a mutant (W610N) that bound maltoheptaose with sufficient affinity to enable structure determination at 2.30 Å resolution. In this structure, maltoheptaose was bound in the active site cleft, allowing us to assign subsites -7 to -1. Moreover, seven oligosaccharide-binding sites were identified on the protein surface, and we postulated that two of these in domain A served as the entrance and exit of the donor/acceptor glucan chains, respectively. Based on these structures, we propose a substrate binding model explaining the mechanism of glycosylation/deglycosylation reactions catalyzed by BE.

Cyanobacterium-Derived Extracellular Carbohydrate Polymer for the Controlled Delivery of Functional Proteins.

The unicellular cyanobacterium Cyanothece sp. CCY 0110 is a highly efficient producer of extracellular polymeric substances (EPS), releasing up to 75% of the polymer to the culture medium. The carbohydrate polymer released to the medium (RPS) was previously isolated and characterized; it is composed of nine different monosaccharides including two uronic acids, and also containing peptides and sulfate groups. Here it is shown that the RPS spontaneously assembles with proteins at high concentrations leading to a phase transition. The proteins are released progressively and structurally intact near physiological conditions, primarily through the swelling of the polymer-protein matrix. The releasing kinetics of the proteins can be modulated through the addition of divalent cations, such as calcium. Notably, the polymer is not toxic to human dermal neonatal fibroblasts in vitro at RPS concentrations bellow 0.1 mg mL-1 . The results show that this polymer is a good candidate for the delivery of therapeutic macromolecules.

Structure of the cyanobactin oxidase ThcOx from Cyanothece sp. PCC 7425, the first structure to be solved at Diamond Light Source beamline I23 by means of S-SAD.

Determination of protein crystal structures requires that the phases are derived independently of the observed measurement of diffraction intensities. Many techniques have been developed to obtain phases, including heavy-atom substitution, molecular replacement and substitution during protein expression of the amino acid methionine with selenomethionine. Although the use of selenium-containing methionine has transformed the experimental determination of phases it is not always possible, either because the variant protein cannot be produced or does not crystallize. Phasing of structures by measuring the anomalous diffraction from S atoms could in theory be almost universal since almost all proteins contain methionine or cysteine. Indeed, many structures have been solved by the so-called native sulfur single-wavelength anomalous diffraction (S-SAD) phasing method. However, the anomalous effect is weak at the wavelengths where data are normally recorded (between 1 and 2 Å) and this limits the potential of this method to well diffracting crystals. Longer wavelengths increase the strength of the anomalous signal but at the cost of increasing air absorption and scatter, which degrade the precision of the anomalous measurement, consequently hindering phase determination. A new instrument, the long-wavelength beamline I23 at Diamond Light Source, was designed to work at significantly longer wavelengths compared with standard synchrotron beamlines in order to open up the native S-SAD method to projects of increasing complexity. Here, the first novel structure, that of the oxidase domain involved in the production of the natural product patellamide, solved on this beamline is reported using data collected to a resolution of 3.15 Šat a wavelength of 3.1 Å. The oxidase is an example of a protein that does not crystallize as the selenium variant and for which no suitable homology model for molecular replacement was available. Initial attempts collecting anomalous diffraction data for native sulfur phasing on a standard macromolecular crystallography beamline using a wavelength of 1.77 Šdid not yield a structure. The new beamline thus has the potential to facilitate structure determination by native S-SAD phasing for what would previously have been regarded as very challenging cases with modestly diffracting crystals and low sulfur content.

Structure and Glycan Binding of a New Cyanovirin-N Homolog.

The HIV-1 envelope glycoprotein gp120 is heavily glycosylated and bears numerous high mannose sugars. These sugars can serve as targets for HIV-inactivating compounds, such as antibodies and lectins, which bind to the glycans and interfere with viral entry into the target cell. We determined the 1.6 Å x-ray structure of Cyt-CVNH, a recently identified lectin from the cyanobacterium Cyanothece(7424), and elucidated its glycan specificity by NMR. The Cyt-CVNH structure and glycan recognition profile are similar to those of other CVNH proteins, with each domain specifically binding to Manα(1-2)Manα units on the D1 and D3 arms of high mannose glycans. However, in contrast to CV-N, no cross-linking and precipitation of the cross-linked species in solution was observed upon Man-9 binding, allowing, for the first time, investigation of the interaction of Man-9 with a member of the CVNH family by NMR. HIV assays showed that Cyt-CVNH is able to inhibit HIV-1 with ∼4-fold higher potency than CV-N(P51G), a stabilized version of wild type CV-N. Therefore, Cyt-CVNH may qualify as a valuable lectin for potential microbicidal use.

Crystal structure of a lipoxygenase from Cyanothece sp. may reveal novel features for substrate acquisition.

In eukaryotes, oxidized PUFAs, so-called oxylipins, are vital signaling molecules. The first step in their biosynthesis may be catalyzed by a lipoxygenase (LOX), which forms hydroperoxides by introducing dioxygen into PUFAs. Here we characterized CspLOX1, a phylogenetically distant LOX family member from Cyanothece sp. PCC 8801 and determined its crystal structure. In addition to the classical two domains found in plant, animal, and coral LOXs, we identified an N-terminal helical extension, reminiscent of the long α-helical insertion in Pseudomonas aeruginosa LOX. In liposome flotation studies, this helical extension, rather than the ß-barrel domain, was crucial for a membrane binding function. Additionally, CspLOX1 could oxygenate 1,2-diarachidonyl-sn-glycero-3-phosphocholine, suggesting that the enzyme may act directly on membranes and that fatty acids bind to the active site in a tail-first orientation. This binding mode is further supported by the fact that CspLOX1 catalyzed oxygenation at the n-10 position of both linoleic and arachidonic acid, resulting in 9R- and 11R-hydroperoxides, respectively. Together these results reveal unifying structural features of LOXs and their function. While the core of the active site is important for lipoxygenation and thus highly conserved, peripheral domains functioning in membrane and substrate binding are more variable.

Crystallization and crystallographic analysis of branching enzymes from Cyanothece sp. ATCC 51142.

Several cyanobacterial species, including Cyanothece sp. ATCC 51142, remarkably have four isoforms of α-glucan branching enzymes (BEs). Based on their primary structures, they are classified into glycoside hydrolase (GH) family 13 (BE1, BE2 and BE3) or family 57 (GH57 BE). In the present study, GH13-type BEs from Cyanothece sp. ATCC 51142 (BE1, BE2 and BE3) have been overexpressed in Escherichia coli and biochemically characterized. The recombinant BE1 was crystallized by the hanging-drop vapour-diffusion method. Crystals of BE1 were obtained at 293 K in the presence of 0.2 M Mg(2+), 7-10%(w/v) ethanol, 0.1 M HEPES-NaOH pH 7.2-7.9. The crystals belonged to the tetragonal space group P41212, with unit-cell parameters a = b = 133.75, c = 185.90 Å, and diffracted to beyond 1.85 Šresolution. Matthews coefficient calculations suggested that the crystals of BE1 contained two molecules in the asymmetric unit.

Antibiotic delivery by liposomes from prokaryotic microorganisms: Similia cum similis works better.

To date the effectiveness of antibiotics is undermined by microbial resistance, threatening public health worldwide. Enhancing the efficacy of the current antibiotic arsenal is an alternative strategy. The administration of antimicrobials encapsulated in nanocarriers, such as liposomes, is considered a viable option, though with some drawbacks related to limited affinity between conventional liposomes and bacterial membranes. Here we propose a novel "top-down" procedure to prepare unconventional liposomes from the membranes of prokaryotes (PD-liposomes). These vectors, being obtained from bacteria with limited growth requirements, also represent low-cost systems for scalable biotechnology production. In depth physico-chemical characterization, carried out with dynamic light scattering (DLS) and Small Angle X-ray Scattering (SAXS), indicated that PD-liposomes can be suitable for the employment as antibiotic vectors. Specifically, DLS showed that the mean diameter of loaded liposomes was ∼200-300nm, while SAXS showed that the structure was similar to conventional liposomes, thus allowing a direct comparison with more standard liposomal formulations. Compared to free penicillin G, PD-liposomes loaded with penicillin G showed minimal inhibitory concentrations against E. coli that were up to 16-times lower. Noteworthy, the extent of the bacterial growth inhibition was found to depend on the microorganisms from which liposomes were derived.

An atypical psbA gene encodes a sentinel D1 protein to form a physiologically relevant inactive photosystem II complex in cyanobacteria.

Photosystem II, a large membrane-bound enzyme complex in cyanobacteria and chloroplasts, mediates light-induced oxidation of water to molecular oxygen. The D1 protein of PSII, encoded by the psbA gene, provides multiple ligands for cofactors crucial to this enzymatic reaction. Cyanobacteria contain multiple psbA genes that respond to various physiological cues and environmental factors. Certain unicellular cyanobacterial cells, such as Cyanothece sp. ATCC 51142, are capable of nitrogen fixation, a highly oxygen-sensitive process, by separating oxygen evolution from nitrogen fixation using a day-night cycle. We have shown that c-psbA4, one of the five psbA orthologs in this cyanobacterium, is exclusively expressed during nighttime. Remarkably, the corresponding D1 isoform has replacements of a number of amino acids that are essential ligands for the catalytic Mn4CaO5 metal center for water oxidation by PSII. At least 30 cyanobacterial strains, most of which are known to have nitrogen fixing abilities, have similar psbA orthologs. We expressed the c-psbA4 gene from Cyanothece 51142 in a 4E-3 mutant strain of the model non-nitrogen-fixing cyanobacterium Synechocystis sp. PCC 6803, which lacks any psbA gene. The resultant strain could not grow photoautotrophically. Moreover, these Synechocystis 6803 cells were incapable of PSII-mediated oxygen evolution. Based on our findings, we have named this physiologically relevant, unusual D1 isoform sentinel D1. Sentinel D1 represents a new class of D1 protein that, when incorporated in a PSII complex, ensures that PSII cannot mediate water oxidation, thus allowing oxygen-sensitive processes such as nitrogen fixation to occur in cyanobacterial cells.

Proteome analyses of strains ATCC 51142 and PCC 7822 of the diazotrophic cyanobacterium Cyanothece sp. under culture conditions resulting in enhanced H2 production.

Cultures of the cyanobacterial genus Cyanothece have been shown to produce high levels of biohydrogen. These strains are diazotrophic and undergo pronounced diurnal cycles when grown under N(2)-fixing conditions in light-dark cycles. We seek to better understand the way in which proteins respond to these diurnal changes, and we performed quantitative proteome analysis of Cyanothece sp. strains ATCC 51142 and PCC 7822 grown under 8 different nutritional conditions. Nitrogenase expression was limited to N(2)-fixing conditions, and in the absence of glycerol, nitrogenase gene expression was linked to the dark period. However, glycerol induced expression of nitrogenase during part of the light period, together with cytochrome c oxidase (Cox), glycogen phosphorylase (Glp), and glycolytic and pentose phosphate pathway (PPP) enzymes. This indicated that nitrogenase expression in the light was facilitated via higher levels of respiration and glycogen breakdown. Key enzymes of the Calvin cycle were inhibited in Cyanothece ATCC 51142 in the presence of glycerol under H(2)-producing conditions, suggesting a competition between these sources of carbon. However, in Cyanothece PCC 7822, the Calvin cycle still played a role in cofactor recycling during H(2) production. Our data comprise the first comprehensive profiling of proteome changes in Cyanothece PCC 7822 and allow an in-depth comparative analysis of major physiological and biochemical processes that influence H(2) production in both strains. Our results revealed many previously uncharacterized proteins that may play a role in nitrogenase activity and in other metabolic pathways and may provide suitable targets for genetic manipulation that would lead to improvement of large-scale H(2) production.

Sustained H(2) production driven by photosynthetic water splitting in a unicellular cyanobacterium.

UNLABELLED: The relationship between dinitrogenase-driven H(2) production and oxygenic photosynthesis was investigated in a unicellular cyanobacterium, Cyanothece sp. ATCC 51142, using a novel custom-built photobioreactor equipped with advanced process control. Continuously illuminated nitrogen-deprived cells evolved H(2) at rates up to 400 µmol ⋅ mg Chl(-1) ⋅ h(-1) in parallel with uninterrupted photosynthetic O(2) production. Notably, sustained coproduction of H(2) and O(2) occurred over 100 h in the presence of CO(2), with both gases displaying inverse oscillations which eventually dampened toward stable rates of 125 and 90 µmol ⋅ mg Chl(-1) ⋅ h(-1), respectively. Oscillations were not observed when CO(2) was omitted, and instead H(2) and O(2) evolution rates were positively correlated. The sustainability of the process was further supported by stable chlorophyll content, maintenance of baseline protein and carbohydrate levels, and an enhanced capacity for linear electron transport as measured by chlorophyll fluorescence throughout the experiment. In situ light saturation analyses of H(2) production displayed a strong dose dependence and lack of O(2) inhibition. Inactivation of photosystem II had substantial long-term effects but did not affect short-term H(2) production, indicating that the process is also supported by photosystem I activity and oxidation of endogenous glycogen. However, mass balance calculations suggest that carbohydrate consumption in the light may, at best, account for no more than 50% of the reductant required for the corresponding H(2) production over that period. Collectively, our results demonstrate that uninterrupted H(2) production in unicellular cyanobacteria can be fueled by water photolysis without the detrimental effects of O(2) and have important implications for sustainable production of biofuels. IMPORTANCE: The study provides an important insight into the photophysiology of light-driven H(2) production by the nitrogen-fixing cyanobacterium Cyanothece sp. strain ATCC 51142. This work is also of significance for biotechnology, supporting the feasibility of "direct biophotolysis." The sustainability of the process, highlighted by prolonged gas evolution with no clear sign of significant decay or apparent photodamage, provides a foundation for the future development of an effective, renewable, and economically efficient bio-H(2) production process.

Crystal structure of cce_0566 from Cyanothece 51142, a protein associated with nitrogen fixation in the DUF269 family.

The crystal structure for cce_0566 (171 aa, 19.4 kDa), a DUF269 annotated protein from the diazotrophic cyanobacterium Cyanothece sp. ATCC 51142, was determined to 1.60Å resolution. Cce_0566 is a homodimer with each molecule composed of eight α-helices folded on one side of a three strand anti-parallel ß-sheet. Hydrophobic interactions between the side chains of largely conserved residues on the surface of each ß-sheet hold the dimer together. The fold observed for cce_0566 may be unique to proteins in the DUF269 family, hence, the protein may also have a function unique to nitrogen fixation. A solvent accessible cleft containing conserved charged residues near the dimer interface could represent the active site or ligand-binding surface for the protein's biological function.

Cloning, expression, crystallization and preliminary crystallographic analysis of a pentapeptide-repeat protein (Rfr23) from the bacterium Cyanothece 51142.

A unique feature of cyanobacteria genomes is the abundance of genes that code for hypothetical proteins containing tandem pentapeptide repeats approximately described by the consensus motif A(N/D)LXX. To date, the structures of two pentapeptide-repeat proteins (PRPs) have been determined, with the tandem pentapeptide-repeat sequences observed to adopt a novel type of right-handed quadrilateral beta-helix, or Rfr-fold, in both structures. One structure, Mycobacterium tuberculosis MfpA, is a 183-residue protein that contains 30 consecutive pentapeptide repeats and appears to offer antibiotic resistance by acting as a DNA mimic. The other structure, Cyanothece 51142 Rfr32, is a 167-residue protein that contains 21 consecutive pentapeptide repeats. The function of Rfr32, like the other 35 hypothetical PRPs identified in the genome of Cyanothece, is unknown. In an effort to understand the role of PRPs in cyanobacteria and to better characterize the structural properties of Rfr-folds with different amino-acid sequences, a second PRP from Cyanothece 51142, Rfr23, has been cloned, expressed and purified. Selenomethione-substituted protein was crystallized by vapor diffusion in hanging drops. Nearly complete SAD and native diffraction data sets were collected from these crystals to 2.5 and 2.1 A resolution, respectively, using synchrotron radiation. The crystals belonged to space group I4(1), with unit-cell parameters a = b = 106.61, c = 53.37 A, and one molecule per asymmetric unit. Preliminary analysis of the electron-density map from the SAD data shows that Rfr23 contains an Rfr-fold.

Characterization of two potentially universal turn motifs that shape the repeated five-residues fold--crystal structure of a lumenal pentapeptide repeat protein from Cyanothece 51142.

The genome of the diurnal cyanobacterium Cyanothece sp. PCC 51142 has recently been sequenced and observed to contain 35 pentapeptide repeat proteins (PRPs). These proteins, while present throughout the prokaryotic and eukaryotic kingdoms, are most abundant in cyanobacteria. The sheer number of PRPs in cyanobacteria coupled with their predicted location in every cellular compartment argues for important, yet unknown, physiological and biochemical functions. To gain biochemical insights, the crystal structure for Rfr32, a 167-residue PRP with an N-terminal 29-residue signal peptide, was determined at 2.1 A resolution. The structure is dominated by 21 tandem pentapeptide repeats that fold into a right-handed quadrilateral beta-helix, or Rfr-fold, as observed for the tandem pentapeptide repeats in the only other PRP structure, the mycobacterial fluoroquinoline resistance protein MfpA from Mycobacterium tuberculosis. Sitting on top of the Rfr-fold are two short, antiparallel alpha-helices, bridged with a disulfide bond, that perhaps prevent edge-to-edge aggregation at the C terminus. Analysis of the main-chain (Phi,Psi) dihedral orientations for the pentapeptide repeats in Rfr32 and MfpA makes it possible to recognize the structural details for the two distinct types of four-residue turns adopted by the pentapeptide repeats in the Rfr-fold. These turns, labeled type II and type IV beta-turns, may be universal motifs that shape the Rfr-fold in all PRPs.