Improved Expression of a Thermostable GH18 Bacterial Chitinase in Two Different Escherichia coli Strains and Its Potential Use in Plant Protection and Biocontrol of Phytopathogenic Fungi.

Chitinases are enzymes that can break down chitin, a major component of the exoskeleton of insects and fungi. This feature makes them potential biopesticides in agriculture since they are considered a safe and environmentally friendly alternative to synthetic pesticides. In this work, we performed a comparative study between two different bacterial expression strains to produce a recombinant chitinase with improved stability.

Role of the phospholipid binding sites, PX of p47 and PB region of Rac1, in the formation of the phagocyte NADPH oxidase complex NOX2.

In phagocytes, superoxide anion (O), the precursor of reactive oxygen species, is produced by the NADPH oxidase complex to kill pathogens. Phagocyte NADPH oxidase consists of the transmembrane cytochrome b (cyt b) and four cytosolic components: p40, p47, p67, and Rac1/2. The phagocyte activation by stimuli leads to activation of signal transduction pathways.

New insights in the molecular regulation of the NADPH oxidase 2 activity: Negative modulation by Poldip2.

Poldip2 was shown to be involved in oxidative signaling to ensure certain biological functions. It was proposed that, in VSMC, by interaction with the Nox4-associated membrane protein p22, Poldip2 stimulates the level of reactive oxygen species (ROS) production. In vitro, with fractionated membranes from HEK393 cells over-expressing Nox4, we confirmed the up-regulation of NADPH oxidase 4 activity by the recombinant and purified Poldip2.

Consequences of the constitutive NOX2 activity in living cells: Cytosol acidification, apoptosis, and localized lipid peroxidation.

The phagocyte NADPH oxidase (NOX2) is a key enzyme of the innate immune system generating superoxide anions (O), precursors of reactive oxygen species. The NOX2 protein complex is composed of six subunits: two membrane proteins (gp91 and p22) forming the catalytic core, three cytosolic proteins (p67, p47 and p40) and a small GTPase Rac. The sophisticated activation mechanism of the NADPH oxidase relies on the assembly of cytosolic subunits with the membrane-bound components.

Accumulation of Cytochrome at the Plasma Membrane: Hallmark of Oxidative Stress in Phagocytic Cells.

Neutrophils play a very key role in the human immune defense against pathogenic infections. The predominant players in this role during the activation of neutrophils are the release of cytotoxic agents stored in the granules and secretory vesicles and the massive production of reactive oxygen species (ROS) initiated by the enzyme NADPH oxidase. In addition, in living organisms, cells are continuously exposed to endogenous (inflammations, elevated neutrophil presence in the vicinity) and exogenous ROS at low and moderate levels (travels by plane, radiotherapy, space irradiation, blood banking, etc.

Mechanistic Insights on Heme-to-Heme Transmembrane Electron Transfer Within NADPH Oxydases From Atomistic Simulations.

NOX5 is a member of the NADPH oxidase family which is dedicated to the production of reactive oxygen species. The molecular mechanisms governing transmembrane electron transfer (ET) that permits to shuttle electrons over the biological membrane have remained elusive for a long time. Using computer simulations, we report conformational dynamics of NOX5 embedded within a realistic membrane environment.

Radiation-induced reactive oxygen species partially assemble neutrophil NADPH oxidase.

Neutrophils are key cells from the innate immune system that destroy invading bacteria or viruses, thanks mainly to the non-mitochondrial reactive oxygen species (ROS) generated by the enzyme NADPH oxidase. Our aim was to study the response of neutrophils to situations of oxidative stress with emphasis on the impact on the NADPH oxidase complex. To mimic oxidative stress, we used gamma irradiation that generated ROS (OH, O and HO) in a quantitative controlled manner.

Impacts of vesicular environment on Nox2 activity measurements in vitro.

Background: The production of superoxide anions (O) by the phagocyte NADPH oxidase complex has a crucial role in the destruction of pathogens in innate immunity. Majority of in vitro studies on the functioning of NADPH oxidase indirectly follows the enzymatic reaction by the superoxide reduction of cytochrome c (cyt c). Only few reports mention the alternative approach consisting in measuring the NADPH consumption rate.

Impact of azole drugs on energetics, kinetics, and ligand migration pathways of CO photo-dissociation in bacterial flavohemoglobins.

Flavohemoglobins (fHbs) are heme proteins found in prokaryotic and eukaryotic microbes. They are involved in NO detoxification through an NO˙ dioxygenase mechanism. The N-terminal heme globin domain allows for binding of gaseous ligands whereas a C-terminal NADH/FADH binding domain facilitates association of redox cofactors necessary for ligand reduction.

Purification and biochemical characterization of a novel thermostable and halotolerant subtilisin SAPN, a serine protease from Melghiribacillus thermohalophilus Nari2A for chitin extraction from crab and shrimp shell by-products.

The present study investigates the purification and biochemical characterization of a novel extracellular serine alkaline protease, subtilisin (called SAPN) from Melghiribacillus thermohalophilus Nari2A. The highest yield of protease (395 IU/g) with white shrimp shell by-product (40 g/L) as a unique source of nutriments in the growth medium was achieved after 52 h at 55 °C. The monomeric enzyme of about 30 kDa was purified to homogeneity by ammonium sulfate fractionation, heat treatment, followed by sequential column chromatographies.

A Close-Up View of the Impact of Arachidonic Acid on the Phagocyte NADPH Oxidase.

The NADPH oxidase NOX2 complex consists of assembled cytosolic and redox membrane proteins. In mammalian cells, natural arachidonic acid (cis-AA), released by activated phospholipase-A2, plays an important role in the activation of the NADPH oxidase, but the mechanism of action of cis-AA is still a matter of debate. In cell-free systems, cis-AA is commonly used for activation although its structural effects are still unclear.

Quinones and nitroaromatic compounds as subversive substrates of Staphylococcus aureus flavohemoglobin.

In microorganisms, flavohemoglobins (FHbs) containing FAD and heme (Fe, metHb) convert NO. into nitrate at the expense of NADH and O. FHbs contribute to bacterial resistance to nitrosative stress.

Phagocyte NADPH oxidase, oxidative stress and lipids: Anti- or pro ageing?

The role of NADPH oxidase in ageing is debated because of the dual roles of free radicals, toxic though necessary. In this paper we summarize some results about two aspects linked to the regulation of the activity of phagocyte NADPH oxidase (Nox2), encountered frequently in elderly people: inflammation and hypercholesterolemia. In the presence of a high amount of reactive oxygen species (ROS) created by itself or by any other source, the enzyme activity is mostly lowered.

Conversion of NOX2 into a constitutive enzyme in vitro and in living cells, after its binding with a chimera of the regulatory subunits.

During the phagocytosis of pathogens by phagocyte cells, the NADPH oxidase complex is activated to produce superoxide anion, a precursor of microbial oxidants. The activated NADPH oxidase complex from phagocytes consists in two transmembrane proteins (Nox2 and p22) and four cytosolic proteins (p40, p47, p67 and Rac1-2). In the resting state of the cells, these proteins are dispersed in the cytosol, the membrane of granules and the plasma membrane.

Functional Assembly of Soluble and Membrane Recombinant Proteins of Mammalian NADPH Oxidase Complex.

Activation of phagocyte cells from an innate immune system is associated with a massive consumption of molecular oxygen to generate highly reactive oxygen species (ROS) as microbial weapons. This is achieved by a multiprotein complex, the so-called NADPH oxidase. The activity of phagocyte NADPH oxidase relies on an assembly of more than five proteins, among them the membrane heterodimer named flavocytochrome b (Cytb ), constituted by the tight association of the gp91 (also named Nox2) and p22 proteins.

The physicochemical properties of membranes correlate with the NADPH oxidase activity.

Background: Phagocytes kill ingested microbes by exposure to high concentrations of toxic reactive species generated by NADPH-oxidases. This membrane-bound electron-transferring enzyme is tightly regulated by cellular signaling cascades. So far, molecular and biophysical studies of the NADPH-oxidase were performed over limited temperature ranges, which weaken our understanding of immune response or inflammatory events.

Titanium Dioxide Nanoparticles Increase Superoxide Anion Production by Acting on NADPH Oxidase.

Titanium dioxide (TiO2) anatase nanoparticles (NPs) are metal oxide NPs commercialized for several uses of everyday life. However their toxicity has been poorly investigated. Cellular internalization of NPs has been shown to activate macrophages and neutrophils that contribute to superoxide anion production by the NADPH oxidase complex.

Contribution of lipid environment to NADPH oxidase activity: influence of sterol.

The NADPH-oxidase complex, which plays beneficial or detrimental role in the inflammatory and degenerative diseases, is a membrane multi-subunit complex tightly regulated in order to produce superoxide anions, precursor of oxygen reactive species (ROS), in cells. The flavocytochrome b(558) (Cytb(558)) is the catalytic core of the NADPH oxidase which consists of two membrane proteins gp91(phox) (highly glycosylated) and p22(phox). In this work we took advantage of heterologous yeast cells engineered to express wild-type bovine Cytb(558) to analyze the properties of the NADPH oxidase activity during the biosynthesis processing steps of gp91(phox) and p22(phox) within endoplasmic reticulum (ER) and plasma membrane (Pmb).

Assembly of phagocyte NADPH oxidase: A concerted binding process?

Background: The phagocyte NADPH-oxidase is a multicomponent enzyme that generates superoxide anions. It comprises a membrane redox component flavocytochrome b558 and four cytosolic proteins (p67(phox), p47(phox), p40(phox) and Rac) that must assemble to produce an active system. In this work we focused on the spatio-temporal control of the activation process of phagocyte NADPH oxidase.

Recombinant form of mammalian gp91(phox) is active in the absence of p22(phox).

The flavocytochrome b558 of the phagocyte NADPH oxidase complex comprises two membrane proteins, a glycosylated gp91phox and a non-glycosylated p22phox. Gp91phox contains all of the redox carriers necessary to reduce molecular oxygen to superoxide using NADPH. The capacity of gp91phox to produce superoxide in the absence of its membrane partner p22phox has been little studied.

Antimicrobial agents act differently on Staphyloccocus aureus and Ralstonia eutropha flavohemoglobins.

Flavohemoglobins (FlavoHb) play a key role in bacterial resistance to nitrosative stress and NO signaling modulation. In this study, we cloned, expressed, and characterized the flavoHb from the opportunistic pathogen, Staphylococcus aureus. The higher amino-acid sequence homology is shared with that from Saccharomyces cerevisiae which was therefore used to build a model structure by homology modeling.

Active site analysis of yeast flavohemoglobin based on its structure with a small ligand or econazole.

Unlabelled: Flavohemoglobins (flavoHbs) serve various microorganisms as the major protective enzymes against NO˙-mediated toxicity. FlavoHbs dominantly function as an NO˙ dioxygenase (O2+ NO→ NO3 -), the required electron being shuttled from NAD(P)H via FAD to the heme iron. The X-ray structures of the flavoHb from Saccharomyces cerevisae presented in complex with an unknown small ligand (Yhb) and with econazole (Yhb(E) ) at 2.