The cyanide-degrading nitrilases are of notable interest for their potential to remediate cyanide contaminated waste streams, especially as generated in the gold mining, pharmaceutical, and ...electroplating industries. This review provides a brief overview of cyanide remediation in general but with a particular focus on the cyanide-degrading nitrilases. These are of special interest as the hydrolysis reaction does not require secondary substrates or cofactors, making these enzymes particularly good candidates for industrial remediation processes. The genetic approaches that have been used to date for engineering improved enzymes are described; however, recent structural insights provide a promising new approach.
Cytochrome P450 reductases (CPRs) are diflavin oxidoreductases that supply electrons to type II cytochrome P450 monooxygenases (CYPs). In addition, it can also reduce other proteins and molecules, ...including cytochrome c, ferricyanide, and different drugs. Although various CPRs have been functionally and structurally characterized, the overall mechanism and its interaction with different redox acceptors remain elusive. One of the main problems regarding electron transfer between CPRs and CYPs is the so-called "uncoupling", whereby NAD(P)H derived electrons are lost due to the reduced intermediates' (FAD and FMN of CPR) interaction with molecular oxygen. Additionally, the decay of the iron-oxygen complex of the CYP can also contribute to loss of reducing equivalents during an unproductive reaction cycle. This phenomenon generates reactive oxygen species (ROS), leading to an inefficient reaction. Here, we present the study of the CPR from Candida tropicalis (CtCPR) lacking the hydrophobic N-terminal part (Δ2-22). The enzyme supports the reduction of cytochrome c and ferricyanide, with an estimated 30% uncoupling during the reactions with cytochrome c. The ROS produced was not influenced by different physicochemical conditions (ionic strength, pH, temperature). The X-ray structures of the enzyme were solved with and without its cofactor, NADPH. Both CtCPR structures exhibited the closed conformation. Comparison with the different solved structures revealed an intricate ionic network responsible for the regulation of the open/closed movement of CtCPR.
The nitrilases are enzymes that convert nitriles to the corresponding acid and ammonia. They are members of a superfamily, which includes amidases and occur in both prokaryotes and eukaryotes. The ...superfamily is characterized by having a homodimeric building block with a αββα-αββα sandwich fold and an active site containing four positionally conserved residues: cys, glu, glu and lys. Their high chemical specificity and frequent enantioselectivity makes them attractive biocatalysts for the production of fine chemicals and pharmaceutical intermediates. Nitrilases are also used in the treatment of toxic industrial effluent and cyanide remediation. The superfamily enzymes have been visualized as dimers, tetramers, hexamers, octamers, tetradecamers, octadecamers and variable length helices, but all nitrilase oligomers have the same basic dimer interface. Moreover, in the case of the octamers, tetradecamers, octadecamers and the helices, common principles of subunit association apply. While the range of industrially interesting reactions catalysed by this enzyme class continues to increase, research efforts are still hampered by the lack of a high resolution microbial nitrilase structure which can provide insights into their specificity, enantioselectivity and the mechanism of catalysis. This review provides an overview of the current progress in elucidation of structure and function in this enzyme class and emphasizes insights that may lead to further biotechnological applications.
Amyloid beta (Aβ) depositions are more abundant in HIV-infected brains. The blood-brain barrier, with its backbone created by endothelial cells, is assumed to be a core player in Aβ homeostasis and ...may contribute to Aβ accumulation in the brain. Exposure to HIV increases shedding of extracellular vesicles (EVs) from human brain endothelial cells and alters EV-Aβ levels. EVs carrying various cargo molecules, including a complex set of proteins, can profoundly affect the biology of surrounding neurovascular unit cells. In the current study, we sought to examine how exposure to HIV, alone or together with Aβ, affects the surface and total proteomic landscape of brain endothelial EVs. By using this unbiased approach, we gained an unprecedented, high-resolution insight into these changes. Our data suggest that HIV and Aβ profoundly remodel the proteome of brain endothelial EVs, altering the pathway networks and functional interactions among proteins. These events may contribute to the EV-mediated amyloid pathology in the HIV-infected brain and may be relevant to HIV-1-associated neurocognitive disorders.
Cyanide-degrading nitrilases in nature Benedik, Michael J.; Sewell, B. Trevor
Journal of general and applied microbiology,
01/2018, Letnik:
64, Številka:
2
Journal Article
A growing literature explores spatial patterns of regional and neighborhood correlates of sexual minority (e.g., lesbian, gay, bisexual) concentration. Such patterns have implications for health and ...wellbeing if there are differences in health-promoting or health-hindering resources in neighborhoods or regions. We conducted a systematic review to assess sexual minority concentration in relation to area unit characteristics.
We included only records published after 1973 and made no exclusions by geography or language. We searched 11 databases (Academic Search Complete, CINAHL, Embase, GeoBase, GeoRef, LGBT Life, PsycINFO, PubMed/MEDLINE, Scopus, Sociological Abstracts, Web of Science) on November 19-21, 2016. We searched reference lists of included records. We used the following inclusion criteria: (1) Record is a quantitative study (that is, it uses statistics to describe or associate two or more variables); (2) Record is about (a) migration or internal migration of, (b) area unit selection by, or (c) concentration of sexual minority people (defined by identity, behavior, or attraction); (3) Criterion 2 is linked to the characteristics of regions or neighborhoods (at any spatial scale).
Dual independent coding resulted in 51 records meeting inclusion criteria from the original pool of 5,591. From these records, we identified the 647 reported results linking sexual minority concentration with area unit characteristics. Of these, 132 were unadjusted relationships between sexual minority concentration and four theory-informed domains of neighborhood influence on health. We identified greater concentration of sexual minorities in regions with more resources and in more urban regions. A limited but troubling literature at the neighborhood level suggested potentially higher concentrations of sexual minorities in neighborhoods with fewer resources.
There are substantial gaps in the literature. We discuss the implications of our findings and gaps in relation to key theories of sexual minority health.
The review was not registered with PROSPERO because it was not eligible for registration at the time of the research project's initiation due to the outcome of interest.
Many large biological macromolecules have inherent structural symmetry, being composed of a few distinct subunits, repeated in a symmetric array. These complexes are often not amenable to traditional ...high-resolution structural determination methods, but can be imaged in functionally relevant states using cryo-electron microscopy (cryo-EM). A number of methods for fitting atomic-scale structures into cryo-EM maps have been developed, including the molecular dynamics flexible fitting (MDFF) method. However, quality and resolution of the cryo-EM map are the major determinants of a method's success. In order to incorporate knowledge of structural symmetry into the fitting procedure, we developed the symmetry-restrained MDFF method. The new method adds to the cryo-EM map-derived potential further restraints on the allowed conformations of a complex during fitting, thereby improving the quality of the resultant structure. The benefit of using symmetry-based restraints during fitting, particularly for medium to low-resolution data, is demonstrated for three different systems.
► Structural symmetry information can be incorporated into flexible fitting ► Symmetry-based restraints improve quality of fitted structures ► Benefits are most pronounced for lower resolution data
Least absolute shrinkage and selection operator (Lasso) regression is a statistical technique that can be used to study the effects of clinical variables in outcome prediction. In this study, we ...aimed at systematically reviewing the application of Lasso regression in gastroenterology for developing predictive models and providing a method of performing Lasso regression. A comprehensive search strategy was conducted in PubMed, Embase and Cochrane CENTRAL databases (
Keywords
: lasso regression; gastrointestinal tract/diseases) following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Studies were screened for eligibility based on pre-defined selection criteria and the data was extracted using a standardized form. Total 16 studies were included, comprising a diverse range of gastroenterological disease-related outcomes. Sample sizes ranged from 134 to 8861 subjects. Eleven studies reported liver disease-related prediction models, while five focused on non-hepatic etiology models. Lasso regression was applied for variable selection, risk prediction and model development, with various validation methods and performance metrics used. Model performance metrics included Area Under the Receiver Operating Characteristics (AUROC), C-index and calibration plots. In gastroenterology, Lasso regression has been used in various diseases such as inflammatory bowel disease, liver disease and esophageal cancer. It is valuable for complex scenarios with many predictors. However, its effectiveness depends on high-quality and complete data. While it identifies important variables, it doesn't provide causal interpretations. Therefore, cautious interpretation is necessary considering the study design and data quality.
Cyanide degrading nitrilases are noted for their potential to detoxify industrial wastewater contaminated with cyanide. However, such application would benefit from an improvement to characteristics ...such as their catalytic activity and stability. Following error-prone PCR for random mutagenesis, several cyanide dihydratase mutants from Bacillus pumilus were isolated based on improved catalysis. Four point mutations, K93R, D172N, A202T, and E327K were characterized and their effects on kinetics, thermostability and pH tolerance were studied. K93R and D172N increased the enzyme's thermostability whereas E327K mutation had a less pronounced effect on stability. The D172N mutation also increased the affinity of the enzyme for its substrate at pH 7.7 but lowered its k cat. However, the A202T mutation, located in the dimerization or the A surface, destabilized the protein and abolished its activity. No significant effect on activity at alkaline pH was observed for any of the purified mutants. These mutations help confirm the model of CynD and are discussed in the context of the protein-protein interfaces leading to the protein quaternary structure.
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