Marta S. P. Carepo

Molybdenum Induces the Expression of a Protein Containing a New Heterometallic Mo-Fe Cluster in DesulfoVibrio alaskensis †

The characterization of a novel Mo-Fe protein (MorP) associated with a system that responds to Mo in Desulfovibrio alaskensis is reported. Biochemical characterization shows that MorP is a periplasmic homomultimer of high molecular weight (260 +/- 13 kDa) consisting of 16-18 monomers of 15321.1 +/- 0.5 Da. The UV/visible absorption spectrum of the as-isolated protein shows absorption peaks around 280, 320, and 570 nm with extinction coefficients of 18700, 12800, and 5000 M(-1) cm(-1), respectively. Metal content, EXAFS data and DFT calculations support the presence of a Mo-2S-[2Fe-2S]-2S-Mo cluster never reported before. Analysis of the available genomes from Desulfovibrio species shows that the MorP encoding gene is located downstream of a sensor and a regulator gene. This type of gene arrangement, called two component system, is used by the cell to regulate diverse physiological processes in response to changes in environmental conditions. Increase of both gene expression and protein production was observed when cells were cultured in the presence of 45 microM molybdenum. Involvement of this system in Mo tolerance of sulfate reducing bacteria is proposed.

NMR assignment of the apo-form of a Desulfovibrio gigas protein containing a novel Mo–Cu cluster

We report the 98% assignment of the apo-form of an orange protein, containing a novel Mo–Cu cluster isolated from Desulfovibrio gigas. This protein presents a region where backbone amide protons exchange fast with bulk solvent becoming undetectable. These residues were assigned using 13C-detection experiments.

Chromobacterium violaceum: important insights for virulence and biotechnological potential by exoproteomic studies.

Chromobacterium violaceum is a beta-proteobacterium with high biotechnological potential, found in tropical environments. This bacterium causes opportunistic infections in both humans and animals, that can spread throughout several tissues, quickly leading to the death of the host. Genomic studies identified potential mechanisms of pathogenicity but no further studies were done to confirm the expression of these systems. In this study 36 unique protein entries were identified in databank from a two-dimensional profile of C. violaceum secreted proteins. Chromobacterium violaceum exoproteomic preliminary studies confirmed the production of proteins identified as virulence factors (such as a collagenase, flagellum proteins, metallopeptidases, and toxins), allowing us to better understand its pathogenicity mechanisms. Biotechnologically interesting proteins (such as chitinase and chitosanase) were also identified among the secreted proteins, as well as proteins involved in the transport and capture of amino acids, carbohydrates, and oxidative stress protection. Overall, the secreted proteins identified provide us important insights on pathogenicity mechanisms, biotechnological potential, and environment adaptation of C. violaceum.

Hydroxyl Radical Generation and DNA Nuclease Activity: A Mechanistic Study Based on a Surface-Immobilized Copper Thioether Clip-Phen Derivative

Coordination compounds of copper have been invoked as major actors in processes involving the reduction of molecular oxygen, mostly with the generation of radical species the assignment for which has, so far, not been fully addressed. In the present work, we have carried out studies in solution and on surfaces to gain insights into the nature of the radical oxygen species (ROS) generated by a copper(II) coordination compound containing a thioether clip-phen derivative, 1,3-bis(1,10-phenanthrolin-2-yloxy)-N-(4-(methylthio)benzylidene)propan-2-amine (2CP-Bz-SMe), enabling its adsorption/immobilization to gold surfaces. Whereas surface plasmon resonance (SPR) and electrochemistry of the adsorbed complex indicated the formation of a dimeric Cu(I) intermediate containing molecular oxygen as a bridging ligand, scanning electrochemical microscopy (SECM) and nuclease assays pointed to the generation of a ROS species. Electron paramagnetic resonance (EPR) data reinforced such conclusions, indicating that radical production was dependent on the amount of oxygen and H2 O2 , thus pointing to a mechanism involving a Fenton-like reaction that results in the production of OH(.) .

De novo synthesis of fatty acids is regulated by FapR protein in Exiguobacterium antarcticum B7, a psychrotrophic bacterium isolated from Antarctica

BackgroundFapR protein from the psychrotrophic species Exiguobacterium antarcticum B7 was expressed and purified, and subsequently evaluated for its capacity to bind to the promoter regions of the fabH1-fabF and fapR-plsX-fabD-fabG operons, using electrophoretic mobility shift assay. The genes that compose these operons encode for enzymes involved in the de novo synthesis of fatty acids molecules. In Bacillus subtilis, FapR regulates the expression of these operons, and consequently has influence in the synthesis of long or short-chain fatty acids. To analyze the bacterial cold adaptation, this is an important metabolic pathway because psychrotrophic microrganisms tend to synthesize short and branched-chain unsaturated fatty acids at cold to maintain cell membrane fluidity.ResultsIn this work, it was observed that recombinant protein was able to bind to the promoter of the fully amplified fabH1-fabF and fapR-plsX-fabD-fabG operons. However, FapR was unable to bind to the promoter of fapR-plsX-fabD-fabG operon when synthesized only up to the protein-binding palindrome 5′-TTAGTACCAGATACTAA-3′, thus showing the importance of the entire promoter sequence for the correct protein-DNA interaction.ConclusionsThrough this observation, we demonstrate that the FapR protein possibly regulates the same operons as described for other species, which emphasizes its importance to cold adaptation process of E. antarcticum B7, a psychrotrophic bacterium isolated at Antarctica.

The heme-based oxygen sensor Rhizobium etli FixL: influence of auxiliary ligands on heme redox potential and implications on the enzyme activity

Conformational changes associated to sensing mechanisms of heme-based protein sensors are a key molecular event that seems to modulate not only the protein activity but also the potential of the FeIII/II redox couple of the heme domain. In this work, midpoint potentials (Em) assigned to the FeIII/II redox couple of the heme domain of FixL from Rhizobium etli (ReFixL) in the unliganded and liganded states were determined by spectroelectrochemistry in the presence of inorganic mediators. In comparison to the unliganded ReFixL protein (+19mV), the binding to ligands that switch off the kinase activity induces a negative shift, i. e. Em=-51, -57 and -156mV for O2, imidazole and CN-, respectively. Upon binding to CO, which does not affect the kinase active, Em was observed at +21mV. The potential values observed for FeIII/II of the heme domain of ReFixL upon binding to CO and O2 do not follow the expected trend based on thermodynamics, assuming that positive potential shift would be expected for ligands that bind to and therefore stabilize the FeII state. Our results suggest that the conformational changes that switch off kinase activity upon O2 binding have knock-on effects to the local environment of the heme, such as solvent rearrangement, destabilize the FeII state and counterbalances the FeII-stabilizing influence of the O2 ligand.

Reconstruction of the Fatty Acid Biosynthetic Pathway of Exiguobacterium antarcticum B7 Based on Genomic and Bibliomic Data

Exiguobacterium antarcticum B7 is extremophile Gram-positive bacteria able to survive in cold environments. A key factor to understanding cold adaptation processes is related to the modification of fatty acids composing the cell membranes of psychrotrophic bacteria. In our study we show the in silico reconstruction of the fatty acid biosynthesis pathway of E. antarcticum B7. To build the stoichiometric model, a semiautomatic procedure was applied, which integrates genome information using KEGG and RAST/SEED. Constraint-based methods, namely, Flux Balance Analysis (FBA) and elementary modes (EM), were applied. FBA was implemented in the sense of hexadecenoic acid production maximization. To evaluate the influence of the gene expression in the fluxome analysis, FBA was also calculated using the log2⁡FC values obtained in the transcriptome analysis at 0°C and 37°C. The fatty acid biosynthesis pathway showed a total of 13 elementary flux modes, four of which showed routes for the production of hexadecenoic acid. The reconstructed pathway demonstrated the capacity of E. antarcticum B7 to de novo produce fatty acid molecules. Under the influence of the transcriptome, the fluxome was altered, promoting the production of short-chain fatty acids. The calculated models contribute to better understanding of the bacterial adaptation at cold environments.

Incorporation of molybdenum in rubredoxin: models for mononuclear molybdenum enzymes

Molybdenum is found in the active site of enzymes usually coordinated by one or two pyranopterin molecules. Here, we mimic an enzyme with a mononuclear molybdenum-bis pyranopterin center by incorporating molybdenum in rubredoxin. In the molybdenum-substituted rubredoxin, the metal ion is coordinated by four sulfurs from conserved cysteine residues of the apo-rubredoxin and two other exogenous ligands, oxygen and thiol, forming a Mo(VI)-(S-Cys)4(O)(X) complex, where X represents –OH or –SR. The rubredoxin molybdenum center is stabilized in a Mo(VI) oxidation state, but can be reduced to Mo(IV) via Mo(V) by dithionite, being a suitable model for the spectroscopic properties of resting and reduced forms of molybdenum-bis pyranopterin-containing enzymes. Preliminary experiments indicate that the molybdenum site built in rubredoxin can promote oxo transfer reactions, as exemplified with the oxidation of arsenite to arsenate.

ArsC3 from Desulfovibrio alaskensis G20, a cation and sulfate-independent highly efficient arsenate reductase

Desulfovibrio alaskensis G20, a sulfate-reducing bacterium, contains an arsRBC2C3 operon that encodes two putative arsenate reductases, DaG20_ArsC2 and DaG20_ArsC3. In this study, resistance assays in E. coli transformed with plasmids containing either of the two recombinant arsenate reductases, showed that only DaG20_ArsC3 is functional and able to confer arsenate resistance. Kinetic studies revealed that this enzyme uses thioredoxin as electron donor and therefore belongs to Staphylococcus aureus plasmid pI258 and Bacillus subtilis thioredoxin-coupled arsenate reductases family. Both enzymes from this family contain a potassium-binding site, but only in Sa_ArsC does potassium actually binds resulting in a lower Km. Important differences between the S. aureus and B. subtilis enzymes and DaG20_ArsC3 are observed. DaG20_ArsC3 contains only two (Asn10, Ser33) of the four (Asn10, Ser33, Thr63, Asp65) conserved amino acid residues that form the potassium-binding site and the kinetics is not significantly affected by the presence of either potassium or sulfate ions. Isothermal titration calorimetry measurements confirmed nonspecific binding of K+ and Na+, corroborating the non-relevance of these cations for catalysis. Furthermore, the low Km and high kcat values determined for DaG20_ArsC3 revealed that this enzyme is the most catalytically efficient potassium-independent arsenate reductase described so far and, for the first time indicates that potassium binding is not essential to have low Km, for Trx-arsenate reductases.

Isolation and characterization of a new Cu-Fe protein from Desulfovibrio aminophilus DSM12254

The isolation and characterization of a new metalloprotein containing Cu and Fe atoms is reported. The as-isolated Cu-Fe protein shows an UV-visible spectrum with absorption bands at 320 nm, 409 nm and 615 nm. Molecular mass of the native protein along with denaturating electrophoresis and mass spectrometry data show that this protein is a multimer consisting of 14+/-1 subunits of 15254.3+/-7.6 Da. Mössbauer spectroscopy data of the as-isolated Cu-Fe protein is consistent with the presence of [2Fe-2S](2+) centers. Data interpretation of the dithionite reduced protein suggest that the metallic cluster could be constituted by two ferromagnetically coupled [2Fe-2S](+) spin delocalized pairs. The biochemical properties of the Cu-Fe protein are similar to the recently reported molybdenum resistance associated protein from Desulfovibrio, D. alaskensis. Furthermore, a BLAST search from the DNA deduced amino acid sequence shows that the Cu-Fe protein has homology with proteins annotated as zinc resistance associated proteins from Desulfovibrio, D. alaskensis, D. vulgaris Hildenborough, D. piger ATCC 29098. These facts suggest a possible role of the Cu-Fe protein in metal tolerance.