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Scientific Reports Oct 2015The central enzyme in the Campylobacter jejuni asparagine-linked glycosylation pathway is the oligosaccharyltransferase (OST), PglB, which transfers preassembled glycans...
The central enzyme in the Campylobacter jejuni asparagine-linked glycosylation pathway is the oligosaccharyltransferase (OST), PglB, which transfers preassembled glycans to specific asparagine residues in target proteins. While C. jejuni PglB (CjPglB) can transfer many diverse glycan structures, the acceptor sites that it recognizes are restricted predominantly to those having a negatively charged residue in the -2 position relative to the asparagine. Here, we investigated the acceptor-site preferences for 23 homologs with natural sequence variation compared to CjPglB. Using an ectopic trans-complementation assay for CjPglB function in glycosylation-competent Escherichia coli, we demonstrated in vivo activity for 16 of the candidate OSTs. Interestingly, the OSTs from Campylobacter coli, Campylobacter upsaliensis, Desulfovibrio desulfuricans, Desulfovibrio gigas, and Desulfovibrio vulgaris, exhibited significantly relaxed specificity towards the -2 position compared to CjPglB. These enzymes glycosylated minimal N-X-T motifs in multiple targets and each followed unique, as yet unknown, rules governing acceptor-site preferences. One notable example is D. gigas PglB, which was the only bacterial OST to glycosylate the Fc domain of human immunoglobulin G at its native 'QYNST' sequon. Overall, we find that a subset of bacterial OSTs follow their own rules for acceptor-site specificity, thereby expanding the glycoengineering toolbox with previously unavailable biocatalytic diversity.
Topics: Amino Acid Motifs; Bacterial Proteins; Computational Biology; Data Mining; Genome, Bacterial; Genomics; Glycoproteins; Glycosylation; Hexosyltransferases; Humans; Immunoglobulin Fc Fragments; Membrane Proteins; Models, Molecular; Molecular Conformation; Phylogeny; Polysaccharides; Polysaccharides, Bacterial; Substrate Specificity; Sweetening Agents
PubMed: 26482295
DOI: 10.1038/srep15237 -
Proceedings of the National Academy of... Jul 2016The six-iron cofactor of [FeFe]-hydrogenases (H-cluster) is the most efficient H2-forming catalyst in nature. It comprises a diiron active site with three carbon...
The six-iron cofactor of [FeFe]-hydrogenases (H-cluster) is the most efficient H2-forming catalyst in nature. It comprises a diiron active site with three carbon monoxide (CO) and two cyanide (CN(-)) ligands in the active oxidized state (Hox) and one additional CO ligand in the inhibited state (Hox-CO). The diatomic ligands are sensitive reporter groups for structural changes of the cofactor. Their vibrational dynamics were monitored by real-time attenuated total reflection Fourier-transform infrared spectroscopy. Combination of (13)CO gas exposure, blue or red light irradiation, and controlled hydration of three different [FeFe]-hydrogenase proteins produced 8 Hox and 16 Hox-CO species with all possible isotopic exchange patterns. Extensive density functional theory calculations revealed the vibrational mode couplings of the carbonyl ligands and uniquely assigned each infrared spectrum to a specific labeling pattern. For Hox-CO, agreement between experimental and calculated infrared frequencies improved by up to one order of magnitude for an apical CN(-) at the distal iron ion of the cofactor as opposed to an apical CO. For Hox, two equally probable isomers with partially rotated ligands were suggested. Interconversion between these structures implies dynamic ligand reorientation at the H-cluster. Our experimental protocol for site-selective (13)CO isotope editing combined with computational species assignment opens new perspectives for characterization of functional intermediates in the catalytic cycle.
Topics: Algal Proteins; Bacterial Proteins; Biocatalysis; Carbon Isotopes; Carbon Monoxide; Chlamydomonas reinhardtii; Clostridium; Cyanides; Desulfovibrio desulfuricans; Hydrogen; Hydrogenase; Iron; Ligands; Spectroscopy, Fourier Transform Infrared
PubMed: 27432985
DOI: 10.1073/pnas.1606178113 -
Proceedings of the National Academy of... Jan 2016The synthesis and assembly of the active site [FeFe] unit of [FeFe]-hydrogenases require at least three maturases. The radical S-adenosyl-l-methionine HydG, the best...
The synthesis and assembly of the active site [FeFe] unit of [FeFe]-hydrogenases require at least three maturases. The radical S-adenosyl-l-methionine HydG, the best characterized of these proteins, is responsible for the synthesis of the hydrogenase CO and CN(-) ligands from tyrosine-derived dehydroglycine (DHG). We speculated that CN(-) and the CO precursor (-):CO2H may be generated through an elimination reaction. We tested this hypothesis with both wild type and HydG variants defective in second iron-sulfur cluster coordination by measuring the in vitro production of CO, CN(-), and (-):CO2H-derived formate. We indeed observed formate production under these conditions. We conclude that HydG is a multifunctional enzyme that produces DHG, CN(-), and CO at three well-differentiated catalytic sites. We also speculate that homocysteine, cysteine, or a related ligand could be involved in Fe(CO)x(CN)y transfer to the HydF carrier/scaffold.
Topics: Carbon Monoxide; Catalysis; Catalytic Domain; Cyanides; Cysteine; Desulfovibrio desulfuricans; Homocysteine; Hydrogenase; Iron-Sulfur Proteins; Ligands; Protein Structure, Secondary; S-Adenosylmethionine; Tyrosine
PubMed: 26699472
DOI: 10.1073/pnas.1515842113 -
New Microbes and New Infections Nov 2019species are anaerobic Gram-negative bacilli that make up part of the human gastrointestinal and vaginal flora. Infection with these bacteria is usually secondary to an...
species are anaerobic Gram-negative bacilli that make up part of the human gastrointestinal and vaginal flora. Infection with these bacteria is usually secondary to an intra-abdominal source. Identification of these bacteria is possible using available contemporary methods. We report the first case of bacteraemia identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).
PubMed: 31763046
DOI: 10.1016/j.nmni.2019.100614 -
Scientific Reports Aug 2017Gaussian network model (GNM), regarded as the simplest and most representative coarse-grained model, has been widely adopted to analyze and reveal protein dynamics and...
Gaussian network model (GNM), regarded as the simplest and most representative coarse-grained model, has been widely adopted to analyze and reveal protein dynamics and functions. Designing a variation of the classical GNM, by defining a new Kirchhoff matrix, is the way to improve the residue flexibility modeling. We combined information arising from local relative solvent accessibility (RSA) between two residues into the Kirchhoff matrix of the parameter-free GNM. The undetermined parameters in the new Kirchhoff matrix were estimated by using particle swarm optimization. The usage of RSA was motivated by the fact that our previous work using RSA based linear regression model resulted out higher prediction quality of the residue flexibility when compared with the classical GNM and the parameter free GNM. Computational experiments, conducted based on one training dataset, two independent datasets and one additional small set derived by molecular dynamics simulations, demonstrated that the average correlation coefficients of the proposed RSA based parameter-free GNM, called RpfGNM, were significantly increased when compared with the parameter-free GNM. Our empirical results indicated that a variation of the classical GNMs by combining other protein structural properties is an attractive way to improve the quality of flexibility modeling.
Topics: Amino Acid Sequence; Amino Acids; Cytochrome c Group; Databases, Protein; Datasets as Topic; Desulfovibrio desulfuricans; Humans; Molecular Dynamics Simulation; Solvents
PubMed: 28790346
DOI: 10.1038/s41598-017-07677-9 -
Genome Announcements Oct 2017Here, we report the draft genome of the Gram-negative, sulfate-reducing bacterium strain G11. Isolated from a rumen fluid enrichment, this culture has been a model...
Here, we report the draft genome of the Gram-negative, sulfate-reducing bacterium strain G11. Isolated from a rumen fluid enrichment, this culture has been a model syntrophic partner due to its metabolic flexibility. The assembly yielded a single circular chromosome of 3,414,943 bp and a 57% G+C content.
PubMed: 29074670
DOI: 10.1128/genomeA.01207-17 -
Environmental Health Perspectives Aug 2014Arsenic (As) toxicity is primarily based on its chemical speciation. Although inorganic and methylated As species are well characterized in terms of metabolism and...
BACKGROUND
Arsenic (As) toxicity is primarily based on its chemical speciation. Although inorganic and methylated As species are well characterized in terms of metabolism and formation in the human body, the origin of thiolated methylarsenicals is still unclear.
OBJECTIVES
We sought to determine whether sulfate-reducing bacteria (SRB) from the human gut are actively involved in the thiolation of monomethylarsonic acid (MMAV).
METHODS
We incubated human fecal and colon microbiota in a batch incubator and in a dynamic gut simulator with a dose of 0.5 mg MMAV in the absence or presence of sodium molybdate, an SRB inhibitor. We monitored the conversion of MMAV into monomethyl monothioarsonate (MMMTAV) and other As species by high-performance liquid chromatography coupled with inductively coupled plasma mass spectrometry analysis. We monitored the sulfate-reducing activity of the SRB by measuring hydrogen sulfide (H2S) production. We used molecular analysis to determine the dominant species of SRB responsible for As thiolation.
RESULTS
In the absence of sodium molybdate, the SRB activity-primarily derived from Desulfovibrio desulfuricans (piger)-was specifically and proportionally correlated (p < 0.01) to MMAV conversion into MMMTAV. Inactivating the SRB with molybdate did not result in MMAV thiolation; however, we observed that the microbiota from a dynamic gut simulator were capable of demethylating 4% of the incubated MMAV into arsenous acid (iAsIII), the trivalent and more toxic form of arsenic acid (iAsV).
CONCLUSION
We found that SRB of human gastrointestinal origin, through their ability to produce H2S, were necessary and sufficient to induce As thiolation. The toxicological consequences of this microbial As speciation change are not yet clear. However, given the efficient epithelial absorption of thiolated methylarsenicals, we conclude that the gut microbiome-and SRB activity in particular-should be incorporated into toxicokinetic analysis carried out after As exposure.
Topics: Arsenic; Arsenicals; Bacteria; Colon; Feces; Gastrointestinal Tract; Humans; Hydrogen Sulfide
PubMed: 24833621
DOI: 10.1289/ehp.1307759 -
Biochimica Et Biophysica Acta Apr 2016The dissimilatory adenosine 5'-phosphosulfate reductase (AprAB) is a key enzyme in the sulfate reduction pathway that catalyzes the reversible two electron reduction of...
The dissimilatory adenosine 5'-phosphosulfate reductase (AprAB) is a key enzyme in the sulfate reduction pathway that catalyzes the reversible two electron reduction of adenosine 5'-phosphosulfate (APS) to sulfite and adenosine monophosphate (AMP). The physiological electron donor for AprAB is proposed to be the QmoABC membrane complex, coupling the quinone-pool to sulfate reduction. However, direct electron transfer between these two proteins has never been observed. In this work we demonstrate for the first time direct electron transfer between the Desulfovibrio desulfuricans ATCC 27774 QmoABC complex and AprAB. Cyclic voltammetry conducted with the modified Qmo electrode and AprAB in the electrolyte solution presented the Qmo electrochemical signature with two additional well-defined one electron redox processes, attributed to the AprAB FAD redox behavior. Moreover, experiments performed under catalytic conditions using the QmoABC modified electrode, with AprAB and APS in solution, show a catalytic current peak develop in the cathodic wave, attributed to substrate reduction, and which is not observed in the absence of QmoABC. Substrate dependence conducted with different electrode preparations (with and without immobilized Qmo) demonstrated that the QmoABC complex is essential for efficient electron delivery to AprAB, in order to sustain catalysis. These results confirm the role of Qmo in electron transfer to AprAB.
Topics: Electrochemistry; Electron Transport; Electron Transport Chain Complex Proteins; Oxidoreductases Acting on Sulfur Group Donors; Quinones
PubMed: 26768116
DOI: 10.1016/j.bbabio.2016.01.001 -
Environmental Science & Technology Jul 2019The potential for inorganic mercury (Hg) to be converted to methylmercury depends, in part, on the chemical form of Hg and its bioavailability to anaerobic...
The potential for inorganic mercury (Hg) to be converted to methylmercury depends, in part, on the chemical form of Hg and its bioavailability to anaerobic microorganisms that can methylate Hg. In anaerobic settings, Hg can be associated with sulfide phases, including ferrous iron sulfide (FeS), which can sorb or be coprecipitated with Hg. The objective of this study was to determine if the aging state of FeS alters the Hg coordination environment as well as the reactivity and bioavailability of sorbed and coprecipitated Hg species. FeS particles were synthesized with and without Hg and aged in anaerobic conditions for multiple time frames spanning from 1 h to 1 month. For FeS particles synthesized without Hg, Hg was subsequently sorbed to the FeS for 1 day. Analysis of Hg speciation of these materials by X-ray absorption near edge spectroscopy revealed a predominance of four-coordinate Hg-S species in the sorbed Hg-FeS solids and a mixture of two- and four-coordinate Hg-S in the coprecipitated Hg-FeS. The leaching potential of the Hg was assessed by exposing the particles to a solution of dissolved glutathione (a thiolate-based Hg chelator). As expected, the sorbed Hg-FeS released more soluble Hg compared to the coprecipitated Hg-FeS. However, when these particles were exposed to Desulfovibrio desulfuricans ND132 (a known Hg methylator), more Hg was methylated from the coprecipitated Hg-FeS than the sorbed Hg-FeS, consistent with expectations from the Hg-S coordination state and inconsistent with the selective leaching results. Overall, these results suggest that the bioavailability of particulate Hg cannot be easily discerned by its leaching potential into bulk solution. Rather, bioavailability entails more subtle interactions at particle-cell interfaces and perhaps correlates with the local Hg-S coordination state in the particles.
Topics: Biological Availability; Iron; Mercury; Methylmercury Compounds; Sulfides
PubMed: 31173690
DOI: 10.1021/acs.est.9b00768 -
Journal of Materials Science. Materials... Sep 2017The susceptibility to the fouling of the NiTi and Ti6Al4V alloys due to the adhesion of microorganisms and the biofilm formation is very significant, especially in the...
The susceptibility to the fouling of the NiTi and Ti6Al4V alloys due to the adhesion of microorganisms and the biofilm formation is very significant, especially in the context of an inflammatory state induced by implants contaminated by bacteria, and the implants corrosion stimulated by bacteria. The aim of this work was to examine the differences between the sulphur-oxidizing bacteria (SOB) and sulphate-reducing bacteria (SRB) strains in their affinity for NiTi and Ti6Al4V alloys. The biofilms formed on alloy surfaces by the cells of five bacterial strains (aerobic SOB Acidithiobacillus thiooxidans and Acidithiobacillus ferrooxidans, and anaerobic SRB Desulfovibrio desulfuricans-3 strains) were studied using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The protein concentrations in liquid media have also been analyzed. The results indicate that both alloys tested may be colonized by SOB and SRB strains. In the initial stage of the biofilm formation, the higher affinity of SRB to both the alloys has been documented. However, the SOB strains have indicated the higher (although differentiated) adaptability to changing environment as compared with SRB. Stimulation of the SRB growth on the alloys surface was observed during incubation in the liquid culture media supplemented with artificial saliva, especially of lower pH (imitated conditions under the inflammatory state, for example in the periodontitis course). The results point to the possible threat to the human health resulting from the contamination of the titanium implant alloys surface by the SOB (A. thiooxidans and A. ferrooxidans) and SRB (D. desulfuricans).
Topics: Acidithiobacillus thiooxidans; Alloys; Bacteria; Bacterial Physiological Phenomena; Biofilms; Desulfovibrio desulfuricans; Humans; Microbial Sensitivity Tests; Nickel; Oxidation-Reduction; Sulfates; Sulfur; Surface Properties; Titanium
PubMed: 28956213
DOI: 10.1007/s10856-017-5988-2