NO synthase: Structures and mechanisms

Daff, Simon

doi:10.1016/j.niox.2010.03.001

Cited by 224 publications

(210 citation statements)

References 128 publications

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“…Our recent HDX-MS studies provided detailed models of the iNOS output state, but, because the FAD/NADPH subdomain was not included in the truncations used in this study, the relative positioning of the entire reductase and oxidase domains could not be determined (7). Based on the crystal structure of the nNOS reductase domain (8), it has been hypothesized that the reductase domains dimerize in the NOS holoenzyme, but this hypothesis remains a point of major uncertainty and contention (6,19,20). Furthermore, despite significant homology, it is unknown whether all three NOS isoforms adopt a similar overall structure.…”

Section: Significancementioning

confidence: 99%

Molecular architecture of mammalian nitric oxide synthases

Campbell

Smith

Potter

et al. 2014

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

122

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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...

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Section: Significancementioning

confidence: 99%

Molecular architecture of mammalian nitric oxide synthases

Campbell

Smith

Potter

et al. 2014

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

122

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“…CaM binding is required for efficient electron transfer from the reductase to the oxygenase domain for •NO production (Alderton et al 2001, Daff 2010. The large conformational change that CaM induces in the reductase domain of the NOS enzymes allows for the FMN domain to interact with both the FAD to accept electrons and pass the electrons on to the heme during catalysis (Welland and Daff 2010).…”

Section: Biological Contextmentioning

confidence: 99%

Chemical shift perturbations induced by residue specific mutations of CaM interacting with NOS peptides

2015

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The regulation of Nitric oxide synthase (NOS) by calmodulin (CaM) plays a major role in a number of key physiological and pathological processes. A detailed molecular level picture of how this regulation is achieved is critical for drug development and for our understanding of protein regulation in general.CaM is a small acidic calcium binding protein and is required to fully activate NOS. The exact mechanism of how CaM activates NOS is not fully understood at this time. Studies have shown CaM to act like a switch that causes a conformational change in NOS to allow for the electron transfer between the reductase and oxygenase domains through a process that is thought to be highly dynamic. The interaction of CaM with NOS is modified by a number of post-translation modifications including phosphorylation. Here we present backbone and sidechain 1 H, 15 N NMR assignments of modified CaM interacting with NOS peptides which provides the basis for a detailed study of CaM-NOS interaction dynamics using 15 N relaxation methods.

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“…NOS, the enzyme that produces NO, is a homodimer with a substrate access channel which extends from the active site toward the dimer interface along with a reductase domain and an oxygenase domain (heme and H4B binding) [16]. In humans, there exist three different isoforms of NOS known as: neuronal NOS (nNOS, NOS1), inducible NOS (iNOS, NOS2), and endothelial NOS (eNOS, NOS3) [17]. All isoforms of NOS utilize L-arginine as a substrate along with oxygen and reduced nicotinamide-adenine-dinucleotide phosphate as co-substrates [18].…”

Section: Physiology Of Vascular Tonementioning

confidence: 99%

Methylene Blue for Distributive Shock: A Potential New Use of an Old Antidote

2013

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Methylene blue is used primarily in the treatment of patients with methemoglobinemia. Most recently, methylene blue has been used as a treatment for refractory distributive shock from a variety of causes such as sepsis and anaphylaxis. Many studies suggest that the nitric oxidecyclic guanosine monophosphate (NO-cGMP) pathway plays a significant role in the pathophysiology of distributive shock. There are some experimental and clinical experiences with the use of methylene blue as a selective inhibitor of the NO-cGMP pathway. Methylene blue may play a role in the treatment of distributive shock when standard treatment fails.

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NO synthase: Structures and mechanisms

Cited by 224 publications

References 128 publications

Molecular architecture of mammalian nitric oxide synthases

Molecular architecture of mammalian nitric oxide synthases

Chemical shift perturbations induced by residue specific mutations of CaM interacting with NOS peptides

Methylene Blue for Distributive Shock: A Potential New Use of an Old Antidote

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