Nitric oxide synthase

Nitric-oxide synthase
Human inducible nitric oxide synthase. PDB 1nsi
Identifiers
EC number	1.14.13.39
CAS number	125978-95-2
Databases
IntEnz	IntEnz view
BRENDA	BRENDA entry
ExPASy	NiceZyme view
KEGG	KEGG entry
MetaCyc	metabolic pathway
PRIAM	profile
PDB structures	RCSB PDB PDBe PDBsum
Gene Ontology	AmiGO / EGO
Search PMC articles PubMed articles NCBI proteins

Nitric oxide synthase, oxygenase domain
Structure of endothelial nitric oxide synthase heme domain.[1]
Identifiers
Symbol	NO_synthase
Pfam	PF02898
InterPro	IPR004030
SCOP	1nos
SUPERFAMILY	1nos
Available protein structures: Pfam structures PDB RCSB PDB; PDBe; PDBj PDBsum structure summary

Nitric oxide synthases (EC 1.14.13.39) (NOSs) are a family of enzymes catalyzing the production of nitric oxide (NO) from L-arginine. NO is an important cellular signaling molecule. It helps modulate vascular tone, insulin secretion, airway tone, and peristalsis, and is involved in angiogenesis and neural development. It may function as a retrograde neurotransmitter. Nitric oxide 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 physiologically relevant concentrations, and produces NO as an immune defense mechanism, as NO is a free radical with an unpaired electron. It is the proximate cause of septic shock and may function in autoimmune disease.

NOS catalyzes the reaction:^[2]

• L-arginine + 3/2 NADPH + 1/2 H⁺ + 2 O₂ $\rightleftharpoons$ citrulline + nitric oxide + 2H₂O + 3/2 NADP⁺

NOS isoforms catalyze other leak and side reactions, such as superoxide production at the expense of NADPH. As such, this stoichiometry is not generally observed, and reflects the three electrons supplied per NO by NADPH.

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.^[3] 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).^[2] 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 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:

• 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.

eNOS plays a critical role in embryonic heart development and morphogenesis of coronary arteries and cardiac valves.^[4]

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 form of nNOS is a major muscle protein that produces signals in response to calcium release from the SR. nNOS in the heart protects against cardiac arrhythmia induced by myocardial infarction.^[5]

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 synthesized 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.

Classification

Different members of the NOS family are encoded by separate genes.^[6] There are three known isoforms in mammals, two are constitutive (cNOS) and the third is inducible (iNOS).^[7] Cloning of NOS enzymes indicates that cNOS include both brain constitutive (NOS1) and endothelial constitutive (NOS3); the third is the inducible (NOS2) gene.^[7] Recently, NOS activity has been demonstrated in several bacterial species, including notorious pathogens Bacillus anthracis and Staphylococcus aureus.^[8]

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

Name	Gene(s)	Location	Function
Neuronal NOS (nNOS or NOS1)	NOS1 (Chromosome 12)	nervous tissue skeletal muscle type II	cell communication
Inducible NOS (iNOS or NOS2) Calcium insensitive	NOS2 (Chromosome 17)	immune system cardiovascular system	immune defense against pathogens
Endothelial NOS (eNOS or NOS3 or cNOS)	NOS3 (Chromosome 7)	endothelium	vasodilation
Bacterial NOS (bNOS)	multiple	various Gram-positive bacteria	defense against oxidative stress, antibiotics, immune attack

nNOS

Neuronal NOS (nNOS) produces NO in nervous tissue in both the central and peripheral nervous system. The gene coding for nNOS is located on Chromosome 12.^[9] 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.^[10]

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.^[11]

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⁺. The gene coding for iNOS is located on Chromosome 17.^[9] 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. iNOS produces large quantities of NO upon stimulation, such as by proinflammatory cytokines (e.g. Interleukin-1, Tumor necrosis factor alpha and Interferon gamma).^[12]

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.^[13]

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.^[14]

eNOS

Endothelial NOS (eNOS), also known as nitric oxide synthase 3 (NOS3), generates NO in blood vessels and is involved with regulating vascular function. The gene coding for eNOS is located on Chromosome 7.^[9] A constitutive Ca²⁺ dependent NOS provides a basal release of NO. eNOS is associated with "caveolae" a component of 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.^[15]

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.^[16][17]

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 N^ω-hydroxy-L-arginine (NOHLA) as an intermediate. 2 mol of O₂ and 1.5 mol of NADPH are consumed per mole of NO formed.^[2]

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 (EC 1.6.2.4) and other flavoproteins. The FMN binding domain is 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.^[18]

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 (EC 1.5.1.33), 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.

Inhibitors

Ronopterin (VAS-203), also known as 4-amino-tetrahydrobiopterin (4-ABH₄), an analogue of BH₄ (a cofactor of NOS), is an NOS inhibitor that is under development as a neuroprotective agent for the treatment of traumatic brain injury. Other NOS inhibitors that have been or are being researched for possible clinical use include cindunistat, A-84643, ONO-1714, L-NOARG, NCX-456, VAS-2381, GW-273629, NXN-462, CKD-712, KD-7040, and guanidinoethyldisulfide, among others.

See also

• Biological functions of nitric oxide
• Nitric-oxide synthase (NAD(P)H-dependent)
• Nitric oxide synthase 2 (inducible)

References

1. PDB: 3N5P​; Delker SL, Xue F, Li H, Jamal J, Silverman RB, Poulos TL (December 2010). "Role of zinc in isoform-selective inhibitor binding to neuronal nitric oxide synthase". Biochemistry 49 (51): 10803–10. doi:10.1021/bi1013479. PMC 3193998. PMID 21138269.
2. Knowles RG, Moncada S (March 1994). "Nitric oxide synthases in mammals". Biochem. J. 298 (2): 249–58. PMC 1137932. PMID 7510950.
3. Liu Q, Gross SS (1996). "Binding sites of nitric oxide synthases". Meth. Enzymol. 268: 311–24. doi:10.1016/S0076-6879(96)68033-1. PMID 8782597.
4. Liu Y, Feng Q (July 2012). "NOing the heart: role of nitric oxide synthase-3 in heart development". Differentiation 84 (1): 54–61. doi:10.1016/j.diff.2012.04.004. PMID 22579300.
5. Burger DE, Lu X, Lei M, Xiang FL, Hammoud L, Jiang M, Wang H, Jones DL, Sims SM, Feng Q (October 2009). "Neuronal nitric oxide synthase protects against myocardial infarction-induced ventricular arrhythmia and mortality in mice". Circulation 120 (14): 1345–54. doi:10.1161/CIRCULATIONAHA.108.846402. PMID 19770398.
6. Taylor BS, Kim YM, Wang Q, Shapiro RA, Billiar TR, Geller DA (November 1997). "Nitric oxide down-regulates hepatocyte-inducible nitric oxide synthase gene expression". Arch Surg 132 (11): 1177–83. doi:10.1001/archsurg.1997.01430350027005. PMID 9366709.
7. Stuehr DJ (May 1999). "Mammalian nitric oxide synthases". Biochim. Biophys. Acta 1411 (2–3): 217–30. doi:10.1016/S0005-2728(99)00016-X. PMID 10320659.
8. Gusarov I, Starodubtseva M, Wang ZQ, McQuade L, Lippard SJ, Stuehr DJ, Nudler E (May 2008). "Bacterial Nitric-oxide Synthases Operate without a Dedicated Redox Partner". J. Biol. Chem. 283 (19): 13140–7. doi:10.1074/jbc.M710178200. PMC 2442334. PMID 18316370.
9. Knowles RG, Moncada S (March 1994). "Nitric oxide synthases in mammals". Biochem. J. 298 (2): 249–58. PMC 1137932. PMID 7510950.
10. Southan GJ, Szabó C (February 1996). "Selective pharmacological inhibition of distinct nitric oxide synthase isoforms". Biochem. Pharmacol. 51 (4): 383–94. doi:10.1016/0006-2952(95)02099-3. PMID 8619882.
11. Ponting CP, Phillips C (March 1995). "DHR domains in syntrophins, neuronal NO synthases and other intracellular proteins". Trends Biochem. Sci. 20 (3): 102–3. doi:10.1016/S0968-0004(00)88973-2. PMID 7535955.
12. Green SJ, Scheller LF, Marletta MA, Seguin MC, Klotz FW, Slayter M, Nelson BJ, Nacy CA (December 1994). "Nitric oxide: cytokine-regulation of nitric oxide in host resistance to intracellular pathogens". Immunol. Lett. 43 (1-2): 87–94. doi:10.1016/0165-2478(94)00158-8. PMID 7537721.
13. Mungrue IN, Husain M, Stewart DJ (October 2002). "The role of NOS in heart failure: lessons from murine genetic models". Heart Fail Rev 7 (4): 407–22. PMID 12379825.
14. Al-Azemi M, Refaat B, Amer S, Ola B, Chapman N, Ledger W (August 2010). "The expression of inducible nitric oxide synthase in the human fallopian tube during the menstrual cycle and in ectopic pregnancy". Fertil. Steril. 94 (3): 833–40. doi:10.1016/j.fertnstert.2009.04.020. PMID 19482272.
15. Liu J, Hughes TE, Sessa WC (June 1997). "The First 35 Amino Acids and Fatty Acylation Sites Determine the Molecular Targeting of Endothelial Nitric Oxide Synthase into the Golgi Region of Cells: A Green Fluorescent Protein Study". J. Cell Biol. 137 (7): 1525–35. doi:10.1083/jcb.137.7.1525. PMC 2137822. PMID 9199168.
16. Gusarov I, Nudler E (September 2005). "NO-mediated cytoprotection: Instant adaptation to oxidative stress in bacteria". Proc. Natl. Acad. Sci. U.S.A. 102 (39): 13855–60. doi:10.1073/pnas.0504307102. PMC 1236549. PMID 16172391.
17. Gusarov I, Shatalin K, Starodubtseva M, Nudler E (September 2009). "Endogenous Nitric Oxide Protects Bacteria Against a Wide Spectrum of Antibiotics". Science 325 (5946): 1380–4. doi:10.1126/science.1175439. PMC 2929644. PMID 19745150.
18. Chinje EC, Stratford IJ (1997). "Role of nitric oxide in growth of solid tumours: a balancing act". Essays Biochem. 32: 61–72. PMID 9493011.

External links

• Nitric oxide synthase at the US National Library of Medicine Medical Subject Headings (MeSH)
• The Nobel Prize in Physiology or Medicine 1998
• University of Edinburgh, School of Chemistry - NO Synthase
• Nitric Oxide Synthase in Proteopedia