Distal lysine (de)coordination in the algal hemoglobin THB1: A combined computer simulation and experimental study.

THB1 is a monomeric truncated hemoglobin from the green alga Chlamydomonas reinhardtii. In the absence of exogenous ligands and at neutral pH, the heme group of THB1 is coordinated by two protein residues, Lys53 and His77. THB1 is thought to function as a nitric oxide dioxygenase, and the distal binding of O2 requires the cleavage of the Fe-Lys53 bond accompanied by protonation and expulsion of the lysine from the heme cavity into the solvent. Nuclear magnetic resonance spectroscopy and crystallographic data have provided dynamic and structural insights of the process, but the details of the mechanism have not been fully elucidated. We applied a combination of computer simulations and site-directed mutagenesis experiments to shed light on this issue. Molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics restrained optimizations were performed to explore the nature of the transition between the decoordinated and lysine-bound states of the ferrous heme in THB1. Lys49 and Arg52, which form ionic interactions with the heme propionates in the X-ray structure of lysine-bound THB1, were observed to assist in maintaining Lys53 inside the protein cavity and play a key role in the transition. Lys49Ala, Arg52Ala and Lys49Ala/Arg52Ala THB1 variants were prepared, and the consequences of the replacements on the Lys (de)coordination equilibrium were characterized experimentally for comparison with computational prediction. The results reinforced the dynamic role of protein-propionate interactions and strongly suggested that cleavage of the Fe-Lys53 bond and ensuing conformational rearrangement is facilitated by protonation of the amino group inside the distal cavity.

Cyanobacteria and Eukaryotic Microalgae as Emerging Sources of Antibacterial Peptides.

Cyanobacteria and microalgae are oxygen-producing photosynthetic unicellular organisms encompassing a great diversity of species, which are able to grow under all types of extreme environments and exposed to a wide variety of predators and microbial pathogens. The antibacterial compounds described for these organisms include alkaloids, fatty acids, indoles, macrolides, peptides, phenols, pigments and terpenes, among others. This review presents an overview of antibacterial peptides isolated from cyanobacteria and microalgae, as well as their synergism and mechanisms of action described so far. Antibacterial cyanopeptides belong to different orders, but mainly from Oscillatoriales and Nostocales. Cyanopeptides have different structures but are mainly cyclic peptides. This vast peptide repertoire includes ribosomal and abundant non-ribosomal peptides, evaluated by standard conventional methodologies against pathogenic Gram-negative and Gram-positive bacteria. The antibacterial activity described for microalgal peptides is considerably scarcer, and limited to protein hydrolysates from two Chlorella species, and few peptides from Tetraselmis suecica. Despite the promising applications of antibacterial peptides and the importance of searching for new natural sources of antibiotics, limitations still persist for their pharmaceutical applications.

Isolation and Characterization of Copper- and Zinc- Binding Metallothioneins from the Marine Alga Ulva compressa (Chlorophyta).

In this work, transcripts encoding three metallothioneins from Ulva compressa (UcMTs) were amplified: The 5'and 3' UTRs by RACE-PCR, and the open reading frames (ORFs) by PCR. Transcripts encoding UcMT1.1 (Crassostrea-like), UcMT2 (Mytilus-like), and UcMT3 (Dreissena-like) showed a 5'UTR of 61, 71, and 65 nucleotides and a 3'UTR of 418, 235, and 193 nucleotides, respectively. UcMT1.1 ORF encodes a protein of 81 amino acids (MW 8.2 KDa) with 25 cysteines (29.4%), arranged as three motifs CC and nine motifs CXC; UcMT2 ORF encode a protein of 90 amino acids (9.05 kDa) with 27 cysteines (30%), arranged as three motifs CC, nine motifs CXC, and one motif CXXC; UcMT3 encode a protein of 139 amino acids (13.4 kDa) with 34 cysteines (24%), arranged as seven motifs CC and seven motifs CXC. UcMT1 and UcMT2 were more similar among each other, showing 60% similarity in amino acids; UcMT3 showed only 31% similarity with UcMT1 and UcMT2. In addition, UcMTs displayed structural similarity with MTs of marine invertebrates MTs and the terrestrial invertebrate Caenorhabtidis elegans MTs, but not with MTs from red or brown macroalgae. The ORFs fused with GST were expressed in bacteria allowing copper accumulation, mainly in MT1 and MT2, and zinc, in the case of the three MTs. Thus, the three MTs allowed copper and zinc accumulation in vivo. UcMTs may play a role in copper and zinc accumulation in U. compressa.

Subunit Asa3 ensures the attachment of the peripheral stalk to the membrane sector of the dimeric ATP synthase of Polytomella sp.

The mitochondrial ATP synthase of Polytomella exhibits a peripheral stalk and a dimerization domain built by the Asa subunits, unique to chlorophycean algae. The topology of these subunits has been extensively studied. Here we explored the interactions of subunit Asa3 using Far Western blotting and subcomplex reconstitution, and found it associates with Asa1 and Asa8. We also identified the novel interactions Asa1-Asa2 and Asa1-Asa7. In silico analyses of Asa3 revealed that it adopts a HEAT repeat-like structure that points to its location within the enzyme based on the available 3D-map of the algal ATP synthase. We suggest that subunit Asa3 is instrumental in securing the attachment of the peripheral stalk to the membrane sector, thus stabilizing the dimeric mitochondrial ATP synthase.

Dissecting the peripheral stalk of the mitochondrial ATP synthase of chlorophycean algae.

The algae Chlamydomonas reinhardtii and Polytomella sp., a green and a colorless member of the chlorophycean lineage respectively, exhibit a highly-stable dimeric mitochondrial F1Fo-ATP synthase (complex V), with a molecular mass of 1600 kDa. Polytomella, lacking both chloroplasts and a cell wall, has greatly facilitated the purification of the algal ATP-synthase. Each monomer of the enzyme has 17 polypeptides, eight of which are the conserved, main functional components, and nine polypeptides (Asa1 to Asa9) unique to chlorophycean algae. These atypical subunits form the two robust peripheral stalks observed in the highly-stable dimer of the algal ATP synthase in several electron-microscopy studies. The topological disposition of the components of the enzyme has been addressed with cross-linking experiments in the isolated complex; generation of subcomplexes by limited dissociation of complex V; detection of subunit-subunit interactions using recombinant subunits; in vitro reconstitution of subcomplexes; silencing of the expression of Asa subunits; and modeling of the overall structural features of the complex by EM image reconstruction. Here, we report that the amphipathic polymer Amphipol A8-35 partially dissociates the enzyme, giving rise to two discrete dimeric subcomplexes, whose compositions were characterized. An updated model for the topological disposition of the 17 polypeptides that constitute the algal enzyme is suggested. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

Functional demonstrations of starch binding domains present in Ostreococcus tauri starch synthases isoforms.

BACKGROUND: Starch-binding domains are key modules present in several enzymes involved in polysaccharide metabolism. These non-catalytic modules have already been described as essential for starch-binding and the catalytic activity of starch synthase III from the higher plant Arabidopsis thaliana. In Ostreococcus tauri, a unicellular green alga of the Prasinophyceae family, there are three SSIII isoforms, known as Ostta SSIII-A, SSIII-B and SSIII-C. RESULTS: In this work, using in silico and in vitro characterization techniques, we have demonstrated that Ostta SSIII-A, SSIII-B and SSIII-C contain two, three and no starch-binding domains, respectively. Additionally, our phylogenetic analysis has indicated that OsttaSSIII-B, presenting three N-terminal SBDs, is the isoform more closely related to higher plant SSIII. Furthermore, the sequence alignment and homology modeling data gathered showed that both the main 3-D structures of all the modeled domains obtained and the main amino acid residues implicated in starch binding are well conserved in O. tauri SSIII starch-binding domains. In addition, adsorption assays showed that OsttaSSIII-A D2 and SSIII-B D2 domains are the two that make the greatest contribution to amylose and amylopectin binding, while OsttaSSIII-B D1 is also important for starch binding. CONCLUSIONS: The results presented here suggest that differences between OsttaSSIII-A and SSIII-B SBDs in the number of and binding of amino acid residues may produce differential affinities for each isoform to polysaccharides. Increasing the knowledge about SBDs may lead to their employment in biomedical and industrial applications.

Marine extremophiles: a source of hydrolases for biotechnological applications.

The marine environment covers almost three quarters of the planet and is where evolution took its first steps. Extremophile microorganisms are found in several extreme marine environments, such as hydrothermal vents, hot springs, salty lakes and deep-sea floors. The ability of these microorganisms to support extremes of temperature, salinity and pressure demonstrates their great potential for biotechnological processes. Hydrolases including amylases, cellulases, peptidases and lipases from hyperthermophiles, psychrophiles, halophiles and piezophiles have been investigated for these reasons. Extremozymes are adapted to work in harsh physical-chemical conditions and their use in various industrial applications such as the biofuel, pharmaceutical, fine chemicals and food industries has increased. The understanding of the specific factors that confer the ability to withstand extreme habitats on such enzymes has become a priority for their biotechnological use. The most studied marine extremophiles are prokaryotes and in this review, we present the most studied archaea and bacteria extremophiles and their hydrolases, and discuss their use for industrial applications.

Algal lectin binding to core (α1-6) fucosylated N-glycans: structural basis for specificity and production of recombinant protein.

We determined the specificity of BTL, a lectin from the red marine alga Bryothamnion triquetrum, toward fucosylated oligosaccharides. BTL showed a strict specificity for the core α1,6-fucosylation, which is an important marker for cancerogenesis and quality control of therapeutical antibodies. The double fucosylation α1,6 and α1,3 was also recognized, but the binding was totally abolished in the sole presence of the α1,3-fucosylation. A more detailed analysis of the specificity of BTL showed a preference for bi- and tri-antennary nonbisected N-glycans. Sialylation or fucosylation at the nonreducing end of N-glycans did not affect the recognition by the lectin. BTL displayed a strong affinity for a core α1,6-fucosylated octasaccharide with a Kd of 12 µM by titration microcalorimetry. The structural characterization of the interaction between BTL and the octasaccharide was obtained by STD-NMR. It demonstrated an extended epitope for recognition that includes the fucose residue, the distal GlcNAc and one mannose residue. Recombinant rBTL was obtained in Escherichia coli and characterized. Its binding properties for carbohydrates were studied using hemagglutination tests and glycan array analysis. rBTL was able to agglutinate rabbit erythrocytes with strong hemagglutination activity only after treatment with papain and trypsin, indicating that its ligands were not directly accessible at the cell surface. The hemagglutinating properties of rBTL confirm the correct folding and functional state of the protein. The results show BTL as a potent candidate for cancer diagnosis and as a reagent for the preparation and quality control of antibodies lacking core α1,6-fucosylated N-glycans.

A proteomic approach to the analysis of the components of the phycobilisomes from two cyanobacteria with complementary chromatic adaptation: Fremyella diplosiphon UTEX B590 and Tolypothrix PCC 7601.

Tolypothrix PCC 7601 and Fremyella diplosiphon UTEX B590 can produce two alternative phycobilisome (PBS) rods. PE-PBSs with one phycocyanin (PC) disk and multiple phycoerythrin (PE) disks are found in cells grown under green light (GL). PC-PBSs with only PC disks are obtained from cells grown under red light (RL). In this manuscript, we show the localization of the linker proteins and ferredoxin-NADP(+) oxidoreductase (FNR) in the PC-PBS and of PE-PBS rods using visible spectroscopy and mass spectrometry. PE-PBSs with different [PE]/[PC] ratios and PC-PBSs with different [PC]/[AP] (AP, allophycocyanin) ratios were isolated. CpeC was the primary rod linker protein found in the PBSs with a [PE]/[PC] ratio of 1.1, which indicates that this is the rod linker at the interphase PC-PE. CpeC and CpeD were identified in the PBSs with a [PE]/[PC] ratio of 1.6, which indicates that CpcD is the linker between the first and the second PE hexamers. Finally, CpeC, CpeD, and CpeE were found in the PBSs with a [PE]/[PC] ratio of 2.9, indicating the position of CpeE between the second and third PE moieties. CpcI2 was identified in the two PC-PBSs obtained from cells grown under RL, which indicates that CpcI2 is the linker between the first and second PC hexamers. CpcH2 was identified only in the PC-PBSs from Tolypothrix with a high [PC]/[AP] ratio of 1.92, which indicates that CpcH2 is the linker between the second and third PC hexamers. The PC-PBSs contained the rod cap protein L(R)(10) (CpcD), but this protein was absent in the PE-PBSs. PE-PBSs (lacking L(R)(10)) incorporated exogenous rFNR in a stoichiometry of up to five FNRs per PBS. A maximum of two FNRs per PBS were found in PC-PBSs (with L(R)(10)). These observations support the hypothesis that FNR binds at the distal ends of the PBS rods in the vacant site of CpcD L(R)(10). Finally, the molecular mass of the core membrane linker (L(CM)) was determined to be 102 kDa from a mass spectrometry analysis.

Bryothamnion seaforthii lectin relaxes vascular smooth muscle: involvement of endothelium and NO synthase.

Bryothamnion seaforthii lectin (BSL) induced reversible concentration-related relaxation of endothelized aorta, maximal at 30 microg/ml (IC(50)= 4.8 +/- 0.6 microg/ml). This effect was inhibited by L-NAME and reversed by mucin, probably via interaction with a specific lectin-binding site on the endothelium activating nitric oxide synthase.

Purification and characterization of a glutathione reductase from Phaeodactylum tricornutum.

Glutathione reductase (E.C.1.8.1.7) was purified from Phaeodactylum tricornutum cells grown axenically in an autotrophic medium. The overall procedure started with preparation of the cell extract and addition of ammonium sulfate to 20% saturation, followed by anion exchange and affinity interaction chromatography (Blue-A- and 2',5'-ADP-Sepharose). Complete purification required native polyacrylamide gel electrophoresis as the final step. The enzyme was purified to homogeneity and functionally characterized. Its native molecular mass was estimated to be 118 kDa; which corresponds to a dimer. The enzyme exhibited a specific activity of 190 U mg(-1) with an optimal activity at pH 8.0 and 32 degrees C. We determined K(m) values of 14 microM and 60 microM for NADPH and oxidized glutathione, respectively. Products inhibited the enzyme according to a hybrid ping-pong reaction mechanism. After MALDI-TOF analysis, the purified enzyme was unambiguously identified as one of the two proteins annotated as glutathione reductases in the genome of the diatom. The properties of the enzyme help to understand redox metabolic scenarios in P. tricornutum.

Modulation of the pharmacological effects of enzymatically-active PLA2 by BTL-2, an isolectin isolated from the Bryothamnion triquetrum red alga.

BACKGROUND: An interaction between lectins from marine algae and PLA2 from rattlesnake was suggested some years ago. We, herein, studied the effects elicited by a small isolectin (BTL-2), isolated from Bryothamnion triquetrum, on the pharmacological and biological activities of a PLA2 isolated from rattlesnake venom (Crotalus durissus cascavella), to better understand the enzymatic and pharmacological mechanisms of the PLA2 and its complex. RESULTS: This PLA2 consisted of 122 amino acids (approximate molecular mass of 14 kDa), its pI was estimated to be 8.3, and its amino acid sequence shared a high degree of similarity with that of other neurotoxic and enzymatically-active PLA2s. BTL-2 had a molecular mass estimated in approximately 9 kDa and was characterized as a basic protein. In addition, BTL-2 did not exhibit any enzymatic activity. The PLA2 and BTL-2 formed a stable heterodimer with a molecular mass of approximately 24-26 kDa, estimated by molecular exclusion HPLC. In the presence of BTL-2, we observed a significant increase in PLA2 activity, 23% higher than that of PLA2 alone. BTL-2 demonstrated an inhibition of 98% in the growth of the Gram-positive bacterial strain, Clavibacter michiganensis michiganensis (Cmm), but only 9.8% inhibition of the Gram-negative bacterial strain, Xanthomonas axonopodis pv passiflorae (Xap). PLA2 decreased bacterial growth by 27.3% and 98.5% for Xap and Cmm, respectively, while incubating these two proteins with PLA2-BTL-2 inhibited their growths by 36.2% for Xap and 98.5% for Cmm.PLA2 significantly induced platelet aggregation in washed platelets, whereas BTL-2 did not induce significant platelet aggregation in any assay. However, BTL-2 significantly inhibited platelet aggregation induced by PLA2. In addition, PLA2 exhibited strong oedematogenic activity, which was decreased in the presence of BTL-2. BTL-2 alone did not induce oedema and did not decrease or abolish the oedema induced by the 48/80 compound. CONCLUSION: The unexpected results observed for the PLA2-BTL-2 complex strongly suggest that the pharmacological activity of this PLA2 is not solely dependent on the presence of enzymatic activity, and that other pharmacological regions may also be involved. In addition, we describe for the first time an interaction between two different molecules, which form a stable complex with significant changes in their original biological action. This opens new possibilities for understanding the function and action of crude venom, an extremely complex mixture of different molecules.