Nitric oxide synthase

Nitric oxide synthases (NOSs) are a family of enzymes that catalyze the production of nitric oxide (NO) from L-arginine. NO is an important cellular signaling molecule, having a vital role in many biological processes. It is the intercellular signal that controls vascular tone (hence blood pressure), insulin secretion, airway tone, and peristalsis, and is involved in angiogenesis (growth of new blood vessels) and in the development of nervous system. It is believed to function as a retrograde neurotransmitter and hence is likely to be important in learning. Nitric oxide signalling is mediated in mammals by the calcium/calmodulin controlled isoenzymes eNOS (endothelial NOS) and nNOS (neuronal NOS); the inducible isoform iNOS is involved in immune response, binds calmodulin at all physiologically relevant concentrations, and produces large amounts of NO as a defense mechanism. It is the proximate cause of septic shock and may play a role in many diseases with an autoimmune etiology.

The canonical reaction catalyzed by NOS is:
 * L-arginine + 3/2 NADPH + H+ + 2 O2 = citrulline + nitric oxide + 3/2 NADP+

NOS isoforms catalyze many other leak and side reactions such as superoxide production at the expense of NADPH, so this stoichiometry is not generally observed. The unusual stoichiometry reflects the three electrons supplied per NO by NADPH; NO is a free radical with an unpaired electron.

NOSs are unusual in that they require five cofactors. Eukaryotic NOS isozymes are catalytically self-sufficient. The electron flow in the NO synthase reaction is: NADPH --> FAD --> FMN --> heme --> O2. Tetrahydrobiopterin provides an additional electron during the catalytic cycle which is replaced during turnover. ). NOS is the only known enzyme that binds flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), heme, tetrahydrobiopterin (BH4) and calmodulin.

Species distribution
Arginine-derived NO synthesis has been identified in mammals, fish, birds, invertebrates, and bacteria. Best studied are mammals, where three distinct genes encode NOS isozymes: neuronal (nNOS or NOS-1), cytokine-inducible (iNOS or NOS-2) and endothelial (eNOS or NOS-3). iNOS and nNOS are soluble and found predominantly in the cytosol, while eNOS is membrane associated. Evidence has been found for NO signaling in plants, but plant genomes are devoid of homologs to the superfamily which generates NO in other kingdoms.

Function
In mammals, the endothelial isoform is the primary signal generator in the control of vascular tone, insulin secretion, and airway tone, is involved in regulation of cardiac function and angiogenesis (growth of new blood vessels). NO produced by eNOS has been shown to be a vasodilator identical to the endothelium-derived relaxing factor ( see nitric oxide (NO) ), produced in response to shear from increased blood flow in arteries. This dilates blood vessels by relaxing smooth muscle in their linings. ENOS is the primary controller of smooth muscle tone. NO activates guanylate cyclase, which induces smooth muscle relaxation by: The neuronal isoform is involved in the development of nervous system. It functions as a retrograde neurotransmitter important in long term potentiation and hence is likely to be important in memory and learning. NNOS has many other physiological functions, including regulation of cardiac function and peristalsis and sexual arousal in males and females. An alternatively spliced from of nNOS is a major muscle protein that produces signals in response to calcium release from the SR. The primary receiver for NO produced by eNOS and nNOS is soluble guanylate cyclase, but many secondary targets have been identified. S-nitrosylation appears to be an important mode of action. The inducible isoform iNOS produces large amounts of NO as a defense mechanism. It is synthisized by many cell types in response to cytokines and is an important factor in the response of the body to attack by parasites, bacterial infection, and tumor growth. It is also the cause of septic shock and may play a role in many diseases with an autoimmune etiology. NOS signaling is involved in development and in fertilization in vertebrates. It has been implicated in transitions between vegetative and reproductive states in invertebrates, and in differentiation leading to spore formation in slime molds. NO produced by bacterial NOS is protective against oxidative damage.
 * Increased intracellular cGMP, which inhibits calcium entry into the cell, and decreases intracellular calcium concentrations
 * Activation of K+ channels, which leads to hyperpolarization and relaxation
 * Stimulates a cGMP-dependent protein kinase that activates myosin light chain phosphatase, the enzyme that dephosphorylates myosin light chains, which leads to smooth muscle relaxation.

Classification
Different members of the NOS family are encoded by separate genes. NOS is one of the most regulated enzymes in biology. There are three known isoforms, two are constitutive (cNOS) and the third is inducible (iNOS). Cloning of NOS enzymes indicates that, cNOS include both brain constitutive (NOS1) and endothelial constitutive (NOS3), the third is the inducible (NOS2) gene. Recently, NOS activity has been demonstrated in several bacterial species, including such notorious pathogens as Bacillus anthracis and Staphylococcus aureus.

The different forms of NO synthase have been classified as follows:

nNOS
Neuronal NOS (nNOS) produces NO in nervous tissue in both the central and peripheral nervous system. Neuronal NOS also performs a role in cell communication and is associated with plasma membranes. nNOS action can be inhibited by NPA (N-propyl-L-arginine). This form of the enzyme is specifically inhibited by 7-nitroindazole.

The subcellular localisation of nNOS in skeletal muscle is mediated by anchoring of nNOS to dystrophin. nNOS contains an additional N-terminal domain, the PDZ domain.

iNOS
As opposed to the critical calcium-dependent regulation of constitutive NOS enzymes (nNOS and eNOS), iNOS has been described as calcium-insensitive, likely due to its tight non-covalent interaction with calmodulin (CaM) and Ca2+. While evidence for ‘baseline’ iNOS expression has been elusive, IRF1 and NF-κB-dependent activation of the inducible NOS promoter supports an inflammation mediated stimulation of this transcript.

From a functional perspective, it is important to recognize that induction of the high-output iNOS usually occurs in an oxidative environment, and thus high levels of NO have the opportunity to react with superoxide leading to peroxynitrite formation and cell toxicity.

These properties may define the roles of iNOS in host immunity, enabling its participation in anti-microbial and anti-tumor activities as part of the oxidative burst of macrophages.

It has been suggested that pathologic generation of nitric oxide through increased iNOS production may decrease tubal ciliary beats and smooth muscle contractions and thus affect embryo transport, which may consequently result in ectopic pregnancy.

eNOS
Endothelial NOS (eNOS), also known as nitric oxide synthase 3 (NOS3), generates NO in blood vessels and is involved with regulating vascular function. A constitutive Ca2+ dependent NOS provides a basal release of NO. eNOS is associated with plasma membranes surrounding cells and the membranes of Golgi bodies within cells. eNOS localisation to endothelial membranes is mediated by cotranslational N-terminal myristoylation and post-translational palmitoylation.

bNOS
Bacterial NOS (bNOS) has been shown to protect bacteria against oxidative stress, diverse antibiotics, and host immune response. bNOS plays a key role in the transcription of superoxide dismutase (SodA). Bacteria late in the log phase who do not possess bNOS fail to upregulate SodA, which disables the defenses against harmful oxidative stress. Initially, bNOS may have been present to prepare the cell for stressful conditions but now seems to help shield the bacteria against conventional antimicrobials. As a clinical application, a bNOS inhibitor could be produced to decrease the load of Gram positive bacteria.

Chemical reaction


Nitric oxide synthases produce NO by catalysing a five-electron oxidation of a guanidino nitrogen of L -arginine ( L -Arg). Oxidation of L -Arg to L -citrulline occurs via two successive monooxygenation reactions producing Nω-hydroxy- L -arginine (NOHLA) as an intermediate. 2 mol of O2 and 1.5 mol of NADPH are consumed per mole of NO formed.

Structure
The enzymes exist as homodimers. In eukaryotes, each monomer consisting of two major regions: an N-terminal oxygenase domain, which belongs to the class of heme-thiolate proteins, and a multi-domain C-terminal reductase, which is homologous to NADPH:cytochrome P450 reductase and other flavoproteins. The FMN binding domain ins homologous to flavodoxins, and the two domain fragment containing the FAD and NADPH binding sites is homologous to flavodoxin-NADPH reductases. The interdomain linker between the oxygenase and reductase domains contains a calmodulin-binding sequence. The oxygenase domain is a unique extended beta sheet cage with binding sites for heme and pterin.

NOSs can be dimeric, calmodulin-dependent or calmodulin-containing cytochrome p450-like hemoprotein that combines reductase and oxygenase catalytic domains in one dimer, bear both flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), and carry out a 5`-electron oxidation of non-aromatic amino acid arginine with the aid of tetrahydrobiopterin.

All three isoforms (each of which is presumed to function as a homodimer during activation) share a carboxyl-terminal reductase domain homologous to the cytochrome P450 reductase. They also share an amino-terminal oxygenase domain containing a heme prosthetic group, which is linked in the middle of the protein to a calmodulin-binding domain. Binding of calmodulin appears to act as a "molecular switch" to enable electron flow from flavin prosthetic groups in the reductase domain to heme. This facilitates the conversion of O2 and L -arginine to NO and L -citrulline. The oxygenase domain of each NOS isoform also contains an BH4 prosthetic group, which is required for the efficient generation of NO. Unlike other enzymes where BH4 is used as a source of reducing equivalents and is recycled by dihydrobiopterin reductase, BH4 activates heme-bound O2 by donating a single electron, which is then recaptured to enable nitric oxide release.

The first nitric oxide synthase to be identified was found in neuronal tissue (NOS1 or nNOS); the endothelial NOS (eNOS or NOS3) was the third to be identified. They were originally classified as "constitutively expressed" and "Ca2+ sensitive" but it is now known that they are present in many different cell types and that expression is regulated under specific physiological conditions.

In NOS1 and NOS3, physiological concentrations of Ca2+ in cells regulate the binding of calmodulin to the "latch domains", thereby initiating electron transfer from the flavins to the heme moieties. In contrast, calmodulin remains tightly bound to the inducible and Ca2+-insensitive isoform (iNOS or NOS2) even at a low intracellular Ca2+ activity, acting essentially as a subunit of this isoform.

Nitric oxide may itself regulate NOS expression and activity. Specifically, NO has been shown to play an important negative feedback regulatory role on NOS3, and therefore vascular endothelial cell function. This process, known formally as S-nitrosation (and referred to by many in the field as S-nitrosylation), has been shown to reversibly inhibit NOS3 activity in vascular endothelial cells. This process may be important because it is regulated by cellular redox conditions and may thereby provide a mechanism for the association between "oxidative stress" and endothelial dysfunction. In addition to NOS3, both NOS1 and NOS2 have been found to be S-nitrosated, but the evidence for dynamic regulation of those NOS isoforms by this process is less complete. In addition, both NOS1 and NOS2 have been shown to form ferrous-nitrosyl complexes in their heme prosthetic groups that may act partially to self-inactivate these enzymes under certain conditions. The rate-limiting step for the production of nitric oxide may well be the availability of L -arginine in some cell types. This may be particularly important after the induction of NOS2.