Search Results (12)

HUG is the HELP-UnaG recombinant fusion protein featuring the typical functions of both HELP and UnaG. In HUG, the HELP domain is a thermoresponsive human elastin-like polypeptide. It forms a shield enwrapping the UnaG domain that emits bilirubin-dependent fluorescence. Here, we recapitulate the technological development of this bifunctional synthetic protein from the theoretical background of its distinct protein moieties to the detailed characterization of its macromolecular and functional properties. These pieces of knowledge are the foundations for HUG production and application in the fluorometric analysis of bilirubin and its congeners, biliverdin and bilirubin glucuronide. These bile pigments are metabolites that arise from the catabolism of heme, the prosthetic group of cytochromes, hemoglobin and several other intracellular enzymes engaged in electron transfer, oxygen transport and protection against oxygen free radicals. The HUG assay is a powerful, user-friendly and affordable analytical tool that alone supports research at each level of complexity or taxonomy of living entities, from enzymology, cell biology and pathophysiology to veterinary and clinical sciences. Full article

Experimental methods of single-molecule enzymology allow scientists to determine physicochemical properties of distinct single molecules of various enzymes and to perform direct monitoring of functioning of enzymes at different steps of their catalytic cycle. The approach based on the use of solid-state nanopores is a promising tool for studying the functioning of single-enzyme molecules. Herein, this approach is employed for monitoring the functioning of cytochrome P450 BM3, which represents a very convenient model of cytochrome P450-containing monooxygenase systems. A nanopore of ~5 nm in diameter has been formed in a 40 nm-thick silicon nitride chip by electron beam drilling (EBD), and a single molecule of the BM3 enzyme has been entrapped in the pore. The functioning of the enzyme molecule has been monitored by recording the time dependence of the ion current through the nanopore during the reaction of laurate hydroxylation. In our experiments, the enzyme molecule has been found to be active for 1500 s. The results of our research can be further used in the development of highly sensitive detectors for single-molecule studies in enzymology. Full article

Chemical studies usually consist of measurements made on large ensembles of molecules with data representing average values for the population. It has been shown that individual molecules of a given enzyme have different properties. Large-scale averaging has in the past masked these differences. Alkaline phosphatase has been used as a model to study this enzyme heterogeneity. The catalytic rates of the individual molecules have been found to differ by over 10-fold, and the activation energy of catalysis by more than two-fold. Differences in properties indicate that differences in structure must exist between the molecules. For alkaline phosphatase, the structural differences have been suggested to be differences in glycosylation, differences due to partial proteolysis, and due to some molecules containing mixtures of active and inactive subunits. The determination of the distribution of activities of populations of this enzyme within a sample has also been shown to be a useful tool in diagnostics. This review discusses the advent of single-molecule enzymology and summarizes its use in the study of alkaline phosphatase using capillary electrophoresis, microscopic well assays, and single-molecule tracking. Full article

The insulin-degrading enzyme (IDE) is a Zn²⁺ peptidase originally discovered as the main enzyme involved in the degradation of insulin and other amyloidogenic peptides, such as the β-amyloid (Aβ) peptide. Therefore, a role for the IDE in the cure of diabetes and Alzheimer’s disease (AD) has been long envisaged. Anyway, its role in degrading amyloidogenic proteins remains not clearly defined and, more recently, novel non-proteolytic functions of the IDE have been proposed. From a structural point of view, the IDE presents an atypical clamshell structure, underscoring unique enigmatic enzymological properties. A better understanding of the structure–function relationship may contribute to solving some existing paradoxes of IDE biology and, in light of its multifunctional activity, might lead to novel therapeutic approaches. Full article

Immunoproteasome is a noncanonical form of proteasome with enzymological properties optimized for the generation of antigenic peptides presented in complex with class I MHC molecules. This enzymatic property makes the modulation of its activity a promising area of research. Nevertheless, immunotherapy has emerged as a front-line treatment of advanced/metastatic tumors providing outstanding improvement of life expectancy, even though not all patients achieve a long-lasting clinical benefit. To enhance the efficacy of the currently available immunotherapies and enable the development of new strategies, a broader knowledge of the dynamics of antigen repertoire processing by cancer cells is needed. Therefore, a better understanding of the role of immunoproteasome in antigen processing and of the therapeutic implication of its modulation is mandatory. Studies on the potential crosstalk between proteasome modulators and immune checkpoint inhibitors could provide novel perspectives and an unexplored treatment option for a variety of cancers. Full article

Isomaltulose is widely used in the food industry as a substitute for sucrose owing to its good processing characteristics and physicochemical properties, which is usually synthesized by sucrose isomerase (SIase) with sucrose as substrate. In this study, a gene pal-2 from Raoultella terrigena was predicted to produce SIase, which was subcloned into pET-28a (+) and transformed to the E. coli system. The purified recombinant SIase Pal-2 was characterized in detail. The enzyme is a monomeric protein with a molecular weight of approximately 70 kDa, showing an optimal temperature of 40 °C and optimal pH value of 5.5. The Michaelis constant (K_m) and maximum reaction rate (V_max) are 62.9 mmol/L and 286.4 U/mg, respectively. The conversion rate of isomaltulose reached the maximum of 81.7% after 6 h with 400 g/L sucrose as the substrate and 25 U/mg sucrose of SIase. Moreover, eight site-directed variants were designed and generated. Compared with the wild-type enzyme, the enzyme activities of two mutants N498P and Q275R were increased by 89.2% and 42.2%, respectively, and the isomaltulose conversion rates of three mutants (Y246L, H287R, and H481P) were improved to 89.1%, 90.7%, and 92.4%, respectively. The work identified a novel SIase from the Raoultella genus and its mutants showed a potential to be used for the production of isomaltulose in the industry. Full article

Silicatein-α is a hydrolase found in siliceous sea sponges with a unique ability to condense and hydrolyse silicon–oxygen bonds. The enzyme is thus of interest from the perspective of its unusual enzymology, and for potential applications in the sustainable synthesis of siloxane-containing compounds. However, research into this enzyme has previously been hindered by the tendency of silicatein-α towards aggregation and insolubility. Herein, we report the development of an improved method for the production of a trigger factor-silicatein fusion protein by switching the previous hexahistidine tag for a Strep-II tag, resulting in 244-fold improvement in protein yield compared to previous methods. Light scattering and thermal denaturation analyses show that under the best storage conditions, although oligomerisation is never entirely abolished, these nanoscale aggregates of the Strep-tagged protein exhibit improved colloidal stability and solubility. Enzymatic assays show that the Strep-tagged protein retains catalytic competency, but exhibits lower activity compared to the His₆-tagged protein. These results suggest that the hexahistidine tag is capable of non-specific catalysis through their imidazole side chains, highlighting the importance of careful consideration when selecting a purification tag. Overall, the Strep-tagged fusion protein reported here can be produced to a higher yield, exhibits greater stability, and allows the native catalytic properties of this protein to be assessed. Full article

Sepsis causes severe dysregulation of organ functions, via the development of oxidative stress and inflammation. These pathophysiological mechanisms are mimicked in mice injected with bacterial lipopolysaccharide (LPS). Here, protective properties of argan oil against LPS-induced oxidative stress and inflammation are explored in the murine model. Mice received standard chow, supplemented with argan oil (AO) or olive oil (OO) for 25 days, before septic shock was provoked with a single intraperitoneal injection of LPS, 16 hours prior to animal sacrifice. In addition to a rise in oxidative stress and inflammatory markers, injected LPS also caused hepatotoxicity, accompanied by hyperglycemia, hypercholesterolemia and hyperuremia. These LPS-associated toxic effects were blunted by AO pretreatment, as corroborated by normal plasma parameters and cell stress markers (glutathione: GSH) and antioxidant enzymology (catalase, CAT; superoxide dismutase, SOD and glutathione peroxidase, GPx). Hematoxylin–eosin staining revealed that AO can protect against acute liver injury, maintaining a normal status, which is pointed out by absent or reduced LPS-induced hepatic damage markers (i.e., alanine aminotransferase (ALT) and aspartate transaminase (AST)). Our work also indicated that AO displayed anti-inflammatory activity, due to down-regulations of genes encoding pro-inflammatory cytokines Interleukin-6 (IL-6) and Tumor Necrosis Factor-α (TNF-α) and in up-regulations of the expression of anti-inflammatory genes encoding Interleukin-4 (IL-4) and Interleukin-10 (IL-10). OO provided animals with similar, though less extensive, protective changes. Collectively our work adds compelling evidence to the protective mechanisms of AO against LPS-induced liver injury and hence therapeutic potentialities, in regard to the management of human sepsis. Activations of IL-4/Peroxisome Proliferator-Activated Receptors (IL-4/PPARs) signaling and, under LPS, an anti-inflammatory IL-10/Liver X Receptor (IL-10/LXR) route, obviously indicated the high potency and plasticity of the anti-inflammatory properties of argan oil. Full article

Derivatives of methylenediphosphonic acid possess wide spectra of biological activities and are used in enzymology as research tools as well as in practical medicine. Carbonyl diphosphonic acid is a promising starting building block for synthesis of functionally substituted methylenediphosphonates. Investigation of the interaction of carbonyl diphosphonic acid with hydroxylamine clearly demonstrates that it is impossible to isolate oxime within the pH range 2–12, while only cyanophosphonic and phosphoric acids are the products of the fast proceeding Beckmann-like fragmentation. In the case of O-alkylhydroxylamines, corresponding alcohols are found in the reaction mixtures in addition to cyanophosphonic and phosphoric acids. Therefore, two residues of phosphonic acid being attached to a carbonyl group provide new properties to this carbonyl group, making its oximes very unstable. This principally differs carbonyl diphosphonic acid from structurally related phosphonoglyoxalic acid and other α-ketophosphonates. Full article

Formylglycine-generating enzymes can selectively recognize and oxidize cysteine residues within the sulfatase sub motif at the terminus of proteins to form aldehyde-bearing formylglycine (FGly) residues, and are normally used in protein labeling. In this study, an aldehyde tag was introduced to proteins using formylglycine-generating enzymes encoded by a reconstructed set of the pET28a plasmid system for enzyme immobilization. The haloacid dehalogenase ST2570 from Sulfolobus tokodaii was used as a model enzyme. The C-terminal aldehyde-tagged ST2570 (ST2570CQ) exhibited significant enzymological properties, such as new free aldehyde groups, a high level of protein expression and improved enzyme activity. SBA-15 has widely been used as an immobilization support for its large surface and excellent thermal and chemical stability. It was functionalized with amino groups by aminopropyltriethoxysilane. The C-terminal aldehyde-tagged ST2570 was immobilized to SBA-15 by covalent binding. The site-specific immobilization of ST2570 avoided the chemical denaturation that occurs in general covalent immobilization and resulted in better fastening compared to physical adsorption. The site-specific immobilized ST2570 showed 3-fold higher thermal stability, 1.2-fold higher catalytic ability and improved operational stability than free ST2570. The site-specific immobilized ST2570 retained 60% of its original activity after seven cycles of batch operation, and it was superior to the ST2570 immobilized to SBA-15 by physical adsorption, which loses 40% of its original activity when used for the second time. It is remarkable that the site-specific immobilized ST2570 still retained 100% of its original activity after 10 cycles of reuse in the semi-continuous flow reactor. Overall, these results provide support for the industrial-scale production and application of site-specific, covalently immobilized ST2570. Full article

Elucidating the origin of enzymatic catalysis stands as one the great challenges of contemporary biochemistry and biophysics. The recent emergence of computational enzymology has enhanced our atomistic-level description of biocatalysis as well the kinetic and thermodynamic properties of their mechanisms. There exists a diversity of computational methods allowing the investigation of specific enzymatic properties. Small or large density functional theory models allow the comparison of a plethora of mechanistic reactive species and divergent catalytic pathways. Molecular docking can model different substrate conformations embedded within enzyme active sites and determine those with optimal binding affinities. Molecular dynamics simulations provide insights into the dynamics and roles of active site components as well as the interactions between substrate and enzymes. Hybrid quantum mechanical/molecular mechanical (QM/MM) can model reactions in active sites while considering steric and electrostatic contributions provided by the surrounding environment. Using previous studies done within our group, on OvoA, EgtB, ThrRS, LuxS and MsrA enzymatic systems, we will review how these methods can be used either independently or cooperatively to get insights into enzymatic catalysis. Full article

Brown algae represent a major component of littoral and sublittoral zones in temperate and subtropical ecosystems. An essential adaptive feature of this independent eukaryotic lineage is the ability to couple oxidative reactions resulting from exposure to sunlight and air with the halogenations of various substrates, thereby addressing various biotic and abiotic stresses i.e., defense against predators, tissue repair, holdfast adhesion, and protection against reactive species generated by oxidative processes. Whereas marine organisms mainly make use of bromine to increase the biological activity of secondary metabolites, some orders of brown algae such as Laminariales have also developed a striking capability to accumulate and to use iodine in physiological adaptations to stress. We review selected aspects of the halogenated metabolism of macrophytic brown algae in the light of the most recent results, which point toward novel functions for iodide accumulation in kelps and the importance of bromination in cell wall modifications and adhesion properties of brown algal propagules. The importance of halogen speciation processes ranges from microbiology to biogeochemistry, through enzymology, cellular biology and ecotoxicology. Full article