Identification of a Flavin Mononucleotide Module Residue Critical for Activity of Inducible Nitrite Oxide Synthase

Liu, Xian De; Mazumdar, Tuhina; Xu, Yi; Getzoff, Elizabeth D.; Eissa, N. Tony

doi:10.4049/jimmunol.0902274

Cited by 6 publications

(7 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Azithromycin when combined with Riboflavin elicits the function of macrophage but lower in comparison to Riboflavin when combined to Fluroquinolone signaling [36]. In our study pre-treatment of macrophages with antibiotics does not lead to residual toxic effects on macrophages, and the effects are due to changes in response to live cells because in our results by FACS analysis of ROS a significant increase in ROS production on addition of antibiotic CIP was documented which cannot be attributed by dead cells.…”

Section: Discussionmentioning

confidence: 46%

Riboflavin along with antibiotics balances reactive oxygen species and inflammatory cytokines and controls Staphylococcus aureus infection by boosting murine macrophage function and regulates inflammation

Dey

Bishayi

2016

J Inflamm

View full text Add to dashboard Cite

BackgroundMacrophages serve as intracellular reservoirs of S. aureus. Recent in vitro studies have confirmed high level resistance by S. aureus to macrophage mediated killing and the intracellular persistence of Staphylococci may play an important role in the pathogenesis. Since this localization protects them from both cell-mediated and humoral immune responses, therefore, a successful anti-staphylococcal therapy should include the elimination of intracellular bacteria, further protecting the host cells from staphylococci-induced cell death. So, only antibiotic therapy may not be helpful, successful therapy needs combination of drugs not only for elimination of pathogen but also for rescuing the host cell for S. aureus induced cell death.MethodsIn keeping with this idea an in vitro study has been done to examine the effect of Riboflavin along with antibiotics on phagocytosis, hydorgen peroxide, superoxide production, antioxidant enzyme levels, and cytokine levels in mouse macrophages for amelioration of the Staphylococcus aureus burden. The immune boosting effects of Riboflavin have been validated through perturbations of redox homeostasis and pro-inflammatory cytokines measurements.ResultsIt was observed that the supplementation of Vitamin B-2 (Riboflavin) not only enhances macrophage function as previously reported but also decreases pro-inflammatory responses in Staphylococcus aureus infected macrophages. The observed influence of Riboflavin on enhanced antimicrobial effects such as enhanced phagocytosis of macrophages exposed to S. aureus, hydrogen peroxide or superoxide production when combined with either ciprofloxacin (CIP) or Azithromycin (AZM) and decrease in pro-inflammatory responses of IFN-γ, IL-6, IL-1β. Riboflavin treatment also decreased NO and TNF-α level possibly by inhibiting the NF-κβ pathway. The increased antioxidant enzymes like glutathione reductase, SOD and GSH level helped in maintaining a stable redox state in the cell.ConclusionRiboflavin plus antibiotic pretreatment not only enhances macrophage functions but also decreases proinflammatory responses in Staphylococcus aureus infected macrophages indicating better bacterial clearance and regulated inflammation which may be considered as a novel and important therapeutic intervention.

show abstract

Section: Discussionmentioning

confidence: 46%

Riboflavin along with antibiotics balances reactive oxygen species and inflammatory cytokines and controls Staphylococcus aureus infection by boosting murine macrophage function and regulates inflammation

Dey

Bishayi

2016

J Inflamm

View full text Add to dashboard Cite

show abstract

“…In that work, a neutralization mutation at the nNOS FMN domain surface was shown to increase NO synthesis activity by enhancing the rate of [Fe(II)−NO] oxidation. Another study showed that FMN domain residue E658 is important for human iNOS activity and also suggested the importance of the FMN module for such activity . These results indicate the importance of appropriate docking of the FMN domain for both efficient IET and substrate−heme interactions.…”

Section: Discussionmentioning

confidence: 88%

Pulsed EPR Determination of the Distance between Heme Iron and FMN Centers in a Human Inducible Nitric Oxide Synthase

et al. 2010

View full text Add to dashboard Cite

Mammalian NOS is a homodimeric flavo-hemoprotein that catalyzes the oxidation of L-arginine to NO. Regulation of NO biosynthesis by NOS is primarily through control of interdomain electron transfer (IET) processes in NOS catalysis. The IET from the FMN to heme domains is essential in the delivery of electrons required for O 2 activation in the heme domain and the subsequent NO synthesis by NOS. The NOS output state for NO production is an IET-competent complex of the FMN-binding domain and heme domain, and thereby it facilitates the IET from the FMN to the catalytic heme site. The structure of the functional output state has not yet been determined. In the absence of crystal structure data for NOS holoenzyme, it is important to experimentally determine the Fe⋯FMN distance to provide a key calibration for computational docking studies and for the IET kinetics studies. Here we used the relaxation-induced dipolar modulation enhancement (RIDME) technique to measure the electron spin echo envelope modulation caused by the dipole interactions between paramagnetic FMN and heme iron centers in the [Fe(III)][FMNH • ] form of a human iNOS oxygenase/FMN bi-domain construct. The FMNH • ⋯Fe distance has been directly determined from the RIDME spectrum. This distance (18.8 ± 0.1 Å) is in excellent agreement with the IET rate constant measured by laser flash photolysis.

show abstract

“…2). The striking detail and recognizable repeating shapes in the 2D averages allowed for immediate assignment of domains based on silhouette and size information provided by known mammalian NOS domain crystal structures (8,(14)(15)(16)21). The central ellipsoid density is the dimerized NOS oxidase domain; the C-shaped density is the FAD/NADPH subdomain; the circular density typically bound within the C-shaped density is the FMN subdomain; the other circular density positioned between the reductase and oxidase domains is CaM bound to the CaM-binding helix of NOS (Fig.…”

Section: Resultsmentioning

confidence: 99%

Molecular architecture of mammalian nitric oxide synthases

Campbell

Smith

Potter

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

122

View full text Add to dashboard Cite

NOSs are homodimeric multidomain enzymes responsible for producing NO. In mammals, NO acts as an intercellular messenger in a variety of signaling reactions, as well as a cytotoxin in the innate immune response. Mammals possess three NOS isoformsinducible, endothelial, and neuronal NOS-that are composed of an N-terminal oxidase domain and a C-terminal reductase domain. Calmodulin (CaM) activates NO synthesis by binding to the helical region connecting these two domains. Although crystal structures of isolated domains have been reported, no structure is available for full-length NOS. We used high-throughput single-particle EM to obtain the structures and higher-order domain organization of all three NOS holoenzymes. The structures of inducible, endothelial, and neuronal NOS with and without CaM bound are similar, consisting of a dimerized oxidase domain flanked by two separated reductase domains. NOS isoforms adopt many conformations enabled by three flexible linkers. These conformations represent snapshots of the continuous electron transfer pathway from the reductase domain to the oxidase domain, which reveal that only a single reductase domain participates in electron transfer at a time, and that CaM activates NOS by constraining rotational motions and by directly binding to the oxidase domain. Direct visualization of these large conformational changes induced during electron transfer provides significant insight into the molecular underpinnings governing NO formation.heme | flavin | electron microscopy | conformational heterogeneity N O has emerged as an integral signaling molecule in biology.NOSs are the only enzymes responsible for NO production in mammals. Disruptions in NO signaling are linked to hypertension, erectile dysfunction, neurodegeneration, stroke, and heart disease (1-3). Three NOS isoforms, which vary slightly in size and composition from 260 to 321 kDa for the NOS homodimer, are present in mammals; each is a distinct gene product differing in subcellular localization, tissue distribution, and mode of regulation. The constitutively expressed NOS isoforms are found primarily in endothelial cells [endothelial NOS (eNOS)] and neuronal cells [neuronal NOS (nNOS)], and NO produced by these isoforms initiates diverse signaling processes, including vasodilation, platelet aggregation, myocardial functions, and neurotransmission (4). The activity of the constitutive NOS isoforms is dependent on intracellular Ca 2+ concentrations. The third isoform, inducible NOS (iNOS), is transcriptionally controlled and produces cytotoxic concentrations of NO at sites of infection or inflammation. Unlike eNOS and nNOS, the activity of iNOS is independent of the intracellular Ca 2+ concentration.Mammalian NOS isoforms use a complex assembly of domains and cofactors to convert L-arginine to L-citrulline and NO, via the intermediate N-hydroxy-L-arginine. Mammalian NOS isoforms are active as homodimers and composed of two primary domains: the N-terminal oxidase domain and the C-terminal reductase domain (Fig. 1A). The re...

show abstract

Identification of a Flavin Mononucleotide Module Residue Critical for Activity of Inducible Nitrite Oxide Synthase

Cited by 6 publications

References 36 publications

Riboflavin along with antibiotics balances reactive oxygen species and inflammatory cytokines and controls Staphylococcus aureus infection by boosting murine macrophage function and regulates inflammation

Riboflavin along with antibiotics balances reactive oxygen species and inflammatory cytokines and controls Staphylococcus aureus infection by boosting murine macrophage function and regulates inflammation

Pulsed EPR Determination of the Distance between Heme Iron and FMN Centers in a Human Inducible Nitric Oxide Synthase

Molecular architecture of mammalian nitric oxide synthases

Contact Info

Product

Resources

About