Domain crossover in the reductase subunit of NADPH-dependent assimilatory sulfite reductase.

NADPH-dependent assimilatory sulfite reductase (SiR) from Escherichia coli performs a six-electron reduction of sulfite to the bioavailable sulfide. SiR is composed of a flavoprotein (SiRFP) reductase subunit and a hemoprotein (SiRHP) oxidase subunit. There is no known high-resolution structure of SiR or SiRFP, thus we do not yet fully understand how the subunits interact to perform their chemistry. Here, we used small-angle neutron scattering to understand the impact of conformationally restricting the highly mobile SiRFP octamer into an electron accepting (closed) or electron donating (open) conformation, showing that SiR remains active, flexible, and asymmetric even with these conformational restrictions. From these scattering data, we model the first solution structure of SiRFP. Further, computational modeling of the N-terminal 52 amino acids that are responsible for SiRFP oligomerization suggests an eight-helical bundle tethers together the SiRFP subunits to form the SiR core. Finally, mass spectrometry analysis of the closed SiRFP variant show that SiRFP is capable of inter-molecular domain crossover, in which the electron donating domain from one polypeptide is able to interact directly with the electron accepting domain of another polypeptide. This structural characterization suggests that SiR performs its high-volume electron transfer through both inter- and intramolecular pathways between SiRFP domains and, thus, cis or trans transfer from reductase to oxidase subunits. Such highly redundant potential for electron transfer makes this system a potential target for designing synthetic enzymes.

Neutron scattering maps the higher-order assembly of NADPH-dependent assimilatory sulfite reductase.

Precursor molecules for biomass incorporation must be imported into cells and made available to the molecular machines that build the cell. Sulfur-containing macromolecules require that sulfur be in its S2- oxidation state before assimilation into amino acids, cofactors, and vitamins that are essential to organisms throughout the biosphere. In α-proteobacteria, NADPH-dependent assimilatory sulfite reductase (SiR) performs the final six-electron reduction of sulfur. SiR is a dodecameric oxidoreductase composed of an octameric flavoprotein reductase (SiRFP) and four hemoprotein metalloenzyme oxidases (SiRHPs). SiR performs the electron transfer reduction reaction to produce sulfide from sulfite through coordinated domain movements and subunit interactions without release of partially reduced intermediates. Efforts to understand the electron transfer mechanism responsible for SiR's efficiency are confounded by structural heterogeneity arising from intrinsically disordered regions throughout its complex, including the flexible linker joining SiRFP's flavin-binding domains. As a result, high-resolution structures of SiR dodecamer and its subcomplexes are unknown, leaving a gap in the fundamental understanding of how SiR performs this uniquely large-volume electron transfer reaction. Here, we use deuterium labeling, in vitro reconstitution, analytical ultracentrifugation (AUC), small-angle neutron scattering (SANS), and neutron contrast variation (NCV) to observe the relative subunit positions within SiR's higher-order assembly. AUC and SANS reveal SiR to be a flexible dodecamer and confirm the mismatched SiRFP and SiRHP subunit stoichiometry. NCV shows that the complex is asymmetric, with SiRHP on the periphery of the complex and the centers of mass between SiRFP and SiRHP components over 100 Å apart. SiRFP undergoes compaction upon assembly into SiR's dodecamer and SiRHP adopts multiple positions in the complex. The resulting map of SiR's higher-order structure supports a cis/trans mechanism for electron transfer between domains of reductase subunits as well as between tightly bound or transiently interacting reductase and oxidase subunits.

Unraveling mechanisms of selenium recovery by facultative anaerobic bacterium Azospira sp. A9D-23B in distinct reactor configurations.

Microbial processes are crucial in the redox transformations of toxic selenium oxyanions. This study focused on isolating an efficient selenate-reducing strain, Azospira sp. A9D-23B, and evaluating its capability to recover extracellular selenium nanoparticles (SeNPs) from selenium-laden wastewater in different reactor setups. Analysis using transmission electron microscopy (TEM) and energy-dispersive X-ray spectroscopy (EDX) revealed significantly higher extracellular SeNPs production (99%) on the biocathode of the bioelectrochemical (BEC) reactor compared to the conventional bioreactor (65%). Further investigations into the selenate reductase activity of strain A9D-23B revealed distinct mechanisms of selenate reduction in BEC and conventional bioreactor settings. Notably, selenate reductases associated with the outer membrane and periplasm displayed higher activity (18.31 ± 3.8 µmol/mg-min) on the BEC reactor's biocathode compared to the upflow anaerobic conventional bioreactor (3.24 ± 2.9 µmol/mg-min). Conversely, the selenate reductases associated with the inner membrane and cytoplasm exhibited lower activity (5.82 ± 2.2 µmol/mg-min) on the BEC reactor's biocathode compared to the conventional bioreactor (9.18 ± 1.6 µmol/mg-min). However, the comparable kinetic parameter ( K m ) across cellular fractions in both reactors suggest that SeNP localization was influenced by enzyme activity rather than selenate affinity. Overall, the mechanism involved in selenate reduction to SeNPs and the strain's efficiency in detoxifying selenate below levels regulated by the U.S. Environmental Protection Agency has broad implications for sustainable environmental remediation strategies.

Dimerization of assimilatory NADPH-dependent sulfite reductase reveals elements for diflavin reductase binding at a minimal interface.

Escherichia coli NADPH-dependent assimilatory sulfite reductase is responsible for fixing sulfur for incorporation into sulfur-containing biomolecules. The oxidoreductase is composed of two subunits, an NADPH, FMN, and FAD-binding diflavin reductase and an iron siroheme and Fe4S4-containing oxidase. How they interact has been an unknown for over 50 years because the complex is highly flexible, thus has been intransigent for traditional X-ray or cryo-EM structural analysis. Using a combination of the chameleon plunging system with a fluorinated lipid we overcame the challenge of preserving the minimal dimer between the subunits for high-resolution cryo-EM analysis. Here, we report the first structure of the complex between the reductase and oxidase, revealing how they interact in a minimal interface. Further, we determined the structural elements that discriminate between the pairing of a siroheme-containing oxidase with a diflavin reductase or a ferredoxin partner to channel the six electrons that reduce sulfite to sulfide.

Contagious diseases and tourism: a systematic review based on bibliometric and content analysis methods.

The present study examines the existing knowledge and intellectual structure on contagious diseases and tourism to map the development of the concept through collaborative networks. Utilising the Scopus and Web of Science bibliometric databases, 328 research records were extracted through keyword searching and forward referencing approaches. Based on these records, the study conducted bibliometric and content analysis to diagnose core themes in the field. The present study's findings are helpful for academia and industry to aid their existing knowledge about contagious diseases, particularly its timeline, geographical spread, and development of coherent themes. A rigorous literature review revealed that the scholarly work in the domain of contagious diseases and tourism revolves around four important themes, namely COVID-19, SARS, Crisis management, and Sustainability. The first theme revolves around COVID-19, highlighting about the impact of COVID-19 on different sectors of the TTH industry, countries, stakeholders, and contexts. Also, researchers foresee COVID-19 as a catalyst to reshape the tourism industry. The next group of studies explained the handling of SARS, particularly by Asian countries. The third cluster elaborated on different stages of crisis and strategies adopted by organizations and countries to manage the crisis. Lastly, a handful of studies in the corpus stated that sustainability in tourism needs to be understood beyond saving the environment and aspiring prosperous travel and tourism industry. The study also suggested the scope of future work.

Comparative Evaluation of the Efficacy of Chlorhexidine, Sodium Hypochlorite, the Diode Laser and Saline in Reducing the Microbial Count in Primary Teeth Root Canals - An In Vivo Study.

Introduction: One of the primary goals of pulpectomy is to decrease the sum total of microorganisms and disinfect the tooth root canal system. To achieve this, mechanical preparation, irrigation, disinfection and obturation of the root canal is necessary. The present study was set out to evaluate the difference in the antibacterial efficacy of primary teeth root canals either irrigated with chlorhexidine, saline, and sodium hypochlorite or irradiated with a soft tissue diode laser (980 nm, Photon Plus, Zolar Tech & Mfg Co. Inc, Ontario, Canada). Methods: Sixty primary teeth of children requiring pulpectomy were divided into 4 groups of 15 each, group 1 (2% chlorhexidine), group 2 (1% sodium hypochlorite), group 3 (laser irradiation) and group 4 (saline). Pulp tissue was extirpated from the canals and the samples were collected using sterile absorbent paper points. After cleaning and shaping, the root canals of the teeth in each group were irrigated using sodium hypochlorite, chlorhexidine, and saline or were irradiated with the laser. The samples were obtained again and sent for microbiological examination. Results: The colony-forming unit (CFU) counts from pre-disinfection sample served as a baseline for comparisons throughout the study. The mean bacterial colony counts of all the isolated bacteria reduced after irrigation or irradiation. Intergroup comparisons showed no significant difference when groups 1, 2, and 3 were compared to each other (P > 0.05). However, a significant difference was seen when groups 1, 2, 3 were compared to group 4 (P < 0.05). Conclusion: Two percent chlorhexidine, 1% sodium hypochlorite and laser irradiation succeeded in reducing the root canal infection. Hence, diode laser irradiation may be a possible supplement to existing protocols for disinfecting the root canal system.