Many proteins depend for their activity on cofactors, associated ions and small molecules. This module contains annotations of processes involved in the synthesis of cofactors, either de novo or from essential molecules consumed in the diet (vitamins), as well as regeneration of active forms of cofactors (Lipmann 1984).
View original pathway at Reactome.
Fujimoto K, Takahashi SY, Katoh S.; ''Mutational analysis of sites in sepiapterin reductase phosphorylated by Ca2+/calmodulin-dependent protein kinase II.''; PubMedEurope PMCScholia
Crabtree MJ, Tatham AL, Hale AB, Alp NJ, Channon KM.; ''Critical role for tetrahydrobiopterin recycling by dihydrofolate reductase in regulation of endothelial nitric-oxide synthase coupling: relative importance of the de novo biopterin synthesis versus salvage pathways.''; PubMedEurope PMCScholia
Bürgisser DM, Thöny B, Redweik U, Hunziker P, Heizmann CW, Blau N.; ''Expression and characterization of recombinant human and rat liver 6-pyruvoyl tetrahydropterin synthase. Modified cysteine residues inhibit the enzyme activity.''; PubMedEurope PMCScholia
Gütlich M, Jaeger E, Rücknagel KP, Werner T, Rödl W, Ziegler I, Bacher A.; ''Human GTP cyclohydrolase I: only one out of three cDNA isoforms gives rise to the active enzyme.''; PubMedEurope PMCScholia
Swick L, Kapatos G.; ''A yeast 2-hybrid analysis of human GTP cyclohydrolase I protein interactions.''; PubMedEurope PMCScholia
Marbois BN, Clarke CF.; ''The COQ7 gene encodes a protein in saccharomyces cerevisiae necessary for ubiquinone biosynthesis.''; PubMedEurope PMCScholia
Vásquez-Vivar J, Martásek P, Whitsett J, Joseph J, Kalyanaraman B.; ''The ratio between tetrahydrobiopterin and oxidized tetrahydrobiopterin analogues controls superoxide release from endothelial nitric oxide synthase: an EPR spin trapping study.''; PubMedEurope PMCScholia
Oess S, Icking A, Fulton D, Govers R, Müller-Esterl W.; ''Subcellular targeting and trafficking of nitric oxide synthases.''; PubMedEurope PMCScholia
Chavan B, Gillbro JM, Rokos H, Schallreuter KU.; ''GTP cyclohydrolase feedback regulatory protein controls cofactor 6-tetrahydrobiopterin synthesis in the cytosol and in the nucleus of epidermal keratinocytes and melanocytes.''; PubMedEurope PMCScholia
Baker TA, Milstien S, Katusic ZS.; ''Effect of vitamin C on the availability of tetrahydrobiopterin in human endothelial cells.''; PubMedEurope PMCScholia
Scherer-Oppliger T, Leimbacher W, Blau N, Thöny B.; ''Serine 19 of human 6-pyruvoyltetrahydropterin synthase is phosphorylated by cGMP protein kinase II.''; PubMedEurope PMCScholia
Pacher P, Beckman JS, Liaudet L.; ''Nitric oxide and peroxynitrite in health and disease.''; PubMedEurope PMCScholia
Tekle M, Turunen M, Dallner G, Chojnacki T, Swiezewska E.; ''Investigation of coenzyme Q biosynthesis in human fibroblast and HepG2 cells.''; PubMedEurope PMCScholia
Geisbrecht BV, Gould SJ.; ''The human PICD gene encodes a cytoplasmic and peroxisomal NADP(+)-dependent isocitrate dehydrogenase.''; PubMedEurope PMCScholia
Takikawa S, Curtius HC, Redweik U, Leimbacher W, Ghisla S.; ''Biosynthesis of tetrahydrobiopterin. Purification and characterization of 6-pyruvoyl-tetrahydropterin synthase from human liver.''; PubMedEurope PMCScholia
Patel KB, Stratford MR, Wardman P, Everett SA.; ''Oxidation of tetrahydrobiopterin by biological radicals and scavenging of the trihydrobiopterin radical by ascorbate.''; PubMedEurope PMCScholia
Dang L, White DW, Gross S, Bennett BD, Bittinger MA, Driggers EM, Fantin VR, Jang HG, Jin S, Keenan MC, Marks KM, Prins RM, Ward PS, Yen KE, Liau LM, Rabinowitz JD, Cantley LC, Thompson CB, Vander Heiden MG, Su SM.; ''Cancer-associated IDH1 mutations produce 2-hydroxyglutarate.''; PubMedEurope PMCScholia
Kone BC, Kuncewicz T, Zhang W, Yu ZY.; ''Protein interactions with nitric oxide synthases: controlling the right time, the right place, and the right amount of nitric oxide.''; PubMedEurope PMCScholia
Lohman DC, Forouhar F, Beebe ET, Stefely MS, Minogue CE, Ulbrich A, Stefely JA, Sukumar S, Luna-Sánchez M, Jochem A, Lew S, Seetharaman J, Xiao R, Wang H, Westphall MS, Wrobel RL, Everett JK, Mitchell JC, López LC, Coon JJ, Tong L, Pagliarini DJ.; ''Mitochondrial COQ9 is a lipid-binding protein that associates with COQ7 to enable coenzyme Q biosynthesis.''; PubMedEurope PMCScholia
Ozeir M, Mühlenhoff U, Webert H, Lill R, Fontecave M, Pierrel F.; ''Coenzyme Q biosynthesis: Coq6 is required for the C5-hydroxylation reaction and substrate analogs rescue Coq6 deficiency.''; PubMedEurope PMCScholia
Jonassen T, Clarke CF.; ''Isolation and functional expression of human COQ3, a gene encoding a methyltransferase required for ubiquinone biosynthesis.''; PubMedEurope PMCScholia
Visser WF, van Roermund CW, Ijlst L, Hellingwerf KJ, Waterham HR, Wanders RJ.; ''First identification of a 2-ketoglutarate/isocitrate transport system in mammalian peroxisomes and its characterization.''; PubMedEurope PMCScholia
Bürgisser DM, Thöny B, Redweik U, Hess D, Heizmann CW, Huber R, Nar H.; ''6-Pyruvoyl tetrahydropterin synthase, an enzyme with a novel type of active site involving both zinc binding and an intersubunit catalytic triad motif; site-directed mutagenesis of the proposed active center, characterization of the metal binding site and modelling of substrate binding.''; PubMedEurope PMCScholia
Nar H, Huber R, Heizmann CW, Thöny B, Bürgisser D.; ''Three-dimensional structure of 6-pyruvoyl tetrahydropterin synthase, an enzyme involved in tetrahydrobiopterin biosynthesis.''; PubMedEurope PMCScholia
Ichinose H, Katoh S, Sueoka T, Titani K, Fujita K, Nagatsu T.; ''Cloning and sequencing of cDNA encoding human sepiapterin reductase--an enzyme involved in tetrahydrobiopterin biosynthesis.''; PubMedEurope PMCScholia
Kuzkaya N, Weissmann N, Harrison DG, Dikalov S.; ''Interactions of peroxynitrite, tetrahydrobiopterin, ascorbic acid, and thiols: implications for uncoupling endothelial nitric-oxide synthase.''; PubMedEurope PMCScholia
Forsgren M, Attersand A, Lake S, Grünler J, Swiezewska E, Dallner G, Climent I.; ''Isolation and functional expression of human COQ2, a gene encoding a polyprenyl transferase involved in the synthesis of CoQ.''; PubMedEurope PMCScholia
Crabtree MJ, Channon KM.; ''Synthesis and recycling of tetrahydrobiopterin in endothelial function and vascular disease.''; PubMedEurope PMCScholia
Heeringa SF, Chernin G, Chaki M, Zhou W, Sloan AJ, Ji Z, Xie LX, Salviati L, Hurd TW, Vega-Warner V, Killen PD, Raphael Y, Ashraf S, Ovunc B, Schoeb DS, McLaughlin HM, Airik R, Vlangos CN, Gbadegesin R, Hinkes B, Saisawat P, Trevisson E, Doimo M, Casarin A, Pertegato V, Giorgi G, Prokisch H, Rötig A, Nürnberg G, Becker C, Wang S, Ozaltin F, Topaloglu R, Bakkaloglu A, Bakkaloglu SA, Müller D, Beissert A, Mir S, Berdeli A, Varpizen S, Zenker M, Matejas V, Santos-Ocaña C, Navas P, Kusakabe T, Kispert A, Akman S, Soliman NA, Krick S, Mundel P, Reiser J, Nürnberg P, Clarke CF, Wiggins RC, Faul C, Hildebrandt F.; ''COQ6 mutations in human patients produce nephrotic syndrome with sensorineural deafness.''; PubMedEurope PMCScholia
Sawabe K, Yamamoto K, Harada Y, Ohashi A, Sugawara Y, Matsuoka H, Hasegawa H.; ''Cellular uptake of sepiapterin and push-pull accumulation of tetrahydrobiopterin.''; PubMedEurope PMCScholia
Casey J, Threlfall DR.; ''Synthesis of 5-demethoxyubiquinone-6 and ubiquinone-6 from 3-hexaprenyl-4-hydroxybenzoate in yeast mitochondria.''; PubMedEurope PMCScholia
Goewert RR, Sippel CJ, Grimm MF, Olson RE.; ''Identification of 3-methoxy-4-hydroxy-5-hexaprenylbenzoic acid as a new intermediate in ubiquinone biosynthesis by Saccharomyces cerevisiae.''; PubMedEurope PMCScholia
Milstien S, Katusic Z.; ''Oxidation of tetrahydrobiopterin by peroxynitrite: implications for vascular endothelial function.''; PubMedEurope PMCScholia
Barkovich RJ, Shtanko A, Shepherd JA, Lee PT, Myles DC, Tzagoloff A, Clarke CF.; ''Characterization of the COQ5 gene from Saccharomyces cerevisiae. Evidence for a C-methyltransferase in ubiquinone biosynthesis.''; PubMedEurope PMCScholia
Gin P, Hsu AY, Rothman SC, Jonassen T, Lee PT, Tzagoloff A, Clarke CF.; ''The Saccharomyces cerevisiae COQ6 gene encodes a mitochondrial flavin-dependent monooxygenase required for coenzyme Q biosynthesis.''; PubMedEurope PMCScholia
Berka V, Yeh HC, Gao D, Kiran F, Tsai AL.; ''Redox function of tetrahydrobiopterin and effect of L-arginine on oxygen binding in endothelial nitric oxide synthase.''; PubMedEurope PMCScholia
Vajo Z, King LM, Jonassen T, Wilkin DJ, Ho N, Munnich A, Clarke CF, Francomano CA.; ''Conservation of the Caenorhabditis elegans timing gene clk-1 from yeast to human: a gene required for ubiquinone biosynthesis with potential implications for aging.''; PubMedEurope PMCScholia
Tran UC, Marbois B, Gin P, Gulmezian M, Jonassen T, Clarke CF.; ''Complementation of Saccharomyces cerevisiae coq7 mutants by mitochondrial targeting of the Escherichia coli UbiF polypeptide: two functions of yeast Coq7 polypeptide in coenzyme Q biosynthesis.''; PubMedEurope PMCScholia
Xu X, Zhao J, Xu Z, Peng B, Huang Q, Arnold E, Ding J.; ''Structures of human cytosolic NADP-dependent isocitrate dehydrogenase reveal a novel self-regulatory mechanism of activity.''; PubMedEurope PMCScholia
Saiki R, Nagata A, Kainou T, Matsuda H, Kawamukai M.; ''Characterization of solanesyl and decaprenyl diphosphate synthases in mice and humans.''; PubMedEurope PMCScholia
Harada T, Kagamiyama H, Hatakeyama K.; ''Feedback regulation mechanisms for the control of GTP cyclohydrolase I activity.''; PubMedEurope PMCScholia
Katoh S, Sueoka T, Yamamoto Y, Takahashi SY.; ''Phosphorylation by Ca2+/calmodulin-dependent protein kinase II and protein kinase C of sepiapterin reductase, the terminal enzyme in the biosynthetic pathway of tetrahydrobiopterin.''; PubMedEurope PMCScholia
Nitric oxide (NO), a multifunctional second messenger, is implicated in physiological processes in mammals that range from immune response and potentiation of synaptic transmission to dilation of blood vessels and muscle relaxation. NO is a highly active molecule that diffuses across cell membranes and cannot be stored inside the producing cell. Its signaling capacity is controlled at the levels of biosynthesis and local availability. Its production by NO synthases is under complex and tight control, being regulated at transcriptional and translational levels, through co- and posttranslational modifications, and by subcellular localization. NO is synthesized from L-arginine by a family of nitric oxide synthases (NOS). Three NOS isoforms have been characterized: neuronal NOS (nNOS, NOS1) primarily found in neuronal tissue and skeletal muscle; inducible NOS (iNOS, NOS2) originally isolated from macrophages and later discovered in many other cell types; and endothelial NOS (eNOS, NOS3) present in vascular endothelial cells, cardiac myocytes, and in blood platelets. The enzymatic activity of all three isoforms is dependent on calmodulin, which binds to nNOS and eNOS at elevated intracellular calcium levels, while it is tightly associated with iNOS even at basal calcium levels. As a result, the enzymatic activity of nNOS and eNOS is modulated by changes in intracellular calcium levels, leading to transient NO production, while iNOS continuously releases NO independent of fluctuations in intracellular calcium levels and is mainly regulated at the gene expression level (Pacher et al. 2007).
The NOS enzymes share a common basic structural organization and requirement for substrate cofactors for enzymatic activity. A central calmodulin-binding motif separates an NH2-terminal oxygenase domain from a COOH-terminal reductase domain. Binding sites for cofactors NADPH, FAD, and FMN are located within the reductase domain, while binding sites for tetrahydrobiopterin (BH4) and heme are located within the oxygenase domain. Once calmodulin binds, it facilitates electron transfer from the cofactors in the reductase domain to heme enabling nitric oxide production. Both nNOS and eNOS contain an additional insert (40-50 amino acids) in the middle of the FMN-binding subdomain that serves as autoinhibitory loop, destabilizing calmodulin binding at low calcium levels and inhibiting electron transfer from FMN to the heme in the absence of calmodulin. iNOS does not contain this insert.
In this Reactome pathway module, details of eNOS activation and regulation are annotated. Originally identified as endothelium-derived relaxing factor, eNOS derived NO is a critical signaling molecule in vascular homeostasis. It regulates blood pressure and vascular tone, and is involved in vascular smooth muscle cell proliferation, platelet aggregation, and leukocyte adhesion. Loss of endothelium derived NO is a key feature of endothelial dysfunction, implicated in the pathogenesis of hypertension and atherosclerosis. The endothelial isoform eNOS is unique among the nitric oxide synthase (NOS) family in that it is co-translationally modified at its amino terminus by myristoylation and is further acylated by palmitoylation (two residues next to the myristoylation site). These modifications target eNOS to the plasma membrane caveolae and lipid rafts.
Factors that stimulate eNOS activation and nitric oxide (NO) production include fluid shear stress generated by blood flow, vascular endothelial growth factor (VEGF), bradykinin, estrogen, insulin, and angiopoietin. The activity of eNOS is further regulated by numerous post-translational modifications, including protein-protein interactions, phosphorylation, and subcellular localization.
Following activation, eNOS shuttles between caveolae and other subcellular compartments such as the noncaveolar plasma membrane portions, Golgi apparatus, and perinuclear structures. This subcellular distribution is variable depending upon cell type and mode of activation.
Subcellular localization of eNOS has a profound effect on its ability to produce NO as the availability of its substrates and cofactors will vary with location. eNOS is primarily particulate, and depending on the cell type, eNOS can be found in several membrane compartments: plasma membrane caveolae, lipid rafts, and intracellular membranes such as the Golgi complex.
The first and rate-limiting enzyme in tetrahydrobiopterin de novo biosynthesis is GTP cyclohydrolase I (GCH1, GTPCHI). Three different isoforms are produced but only isoform 1 is functionally active (Gütlich et al. 1994). GCH1 is functional as a homodecamer. First, a monomer of GCH1 forms a dimer. Then five dimers arrange into a ring-like structure to form the homodecamer (Nar et al. 1995).
High levels of the end product, BH4, negatively regulates GCH1. It does this via GTP cyclohydrolase 1 feedback regulatory protein (GCHFR). BH4-dependant GCHFR in the form of a homopentamer complexes with the decameric GCH1 enzyme in the ratio 2:1 to inactivate it. L-phenylalanine reverses this inhibition. These regulatory steps control the biosynthesis of BH4. (Swick & Kapatos 2006, Chavan et al. 2006, Harada et al. 1993).
6-pyruvoyl tetrahydrobiopterin synthase (PTPS) (Takikawa et al. 1986) catalyses the second step in BH4 biosynthesis, the dephosphorylation of DHNTP to 6-pyruvoyl-tetrahydropterin (PTHP). PTPS is believed to function as a homohexamer (Nar et al. 1994, Bürgisser et al. 1994) and has a requirement for Zn2+ (one Zn2+ ion bound per subunit) and Mg2+ ions for activity (Bürgisser et al. 1995). The phosphorylation of Ser-19 is an essential modification for enzyme activity (Scherer-Oppliger et al. 1999).
The cofactor tetrahydrobiopterin (BH4) ensures endothelial nitric oxide synthase (eNOS) couples electron transfer to L-arginine oxidation (Berka et al. 2004). During catalysis, electrons derived from NADPH transfer to the flavins FAD and FMN in the reductase domain of eNOS and then on to the ferric heme in the oxygenase domain of eNOS. BH4 can donate an electron to intermediates in this electron transfer and is oxidised in the process, forming the BH3 radical. This radical can be reduced back to BH4 by iron, completing the cycle and forming ferrous iron again. Heme reduction enables O2 binding and L-arginine oxidation to occur within the oxygenase domain (Stuehr et al. 2009).
In the second salvage step, dihydrofolate reductase (DHFR) can regenerate BH4 from BH2, a process which increases the BH4:BH2 ratio providing BH4 for coupled eNOS production of NO. In mice cell lines, DHFR inhibition or knockdown diminishes the BH4:BH2 ratio and exacerbates eNOS uncoupling (Crabtree et al. 2009).
The oxidation product of BH4, 7,8-dihydrobiopterin (BH2), can compete with BH4 for binding to eNOS. This can lead to the uncoupling of eNOS and can result in the formation of reactive oxygen species (Vasquez-Vivar et al. 2002).
To become active, sepiapterin reductase (SPR) must first be phosphorylated (serine 213 in humans) by Ca2+/calmodulin-dependent protein kinase II (Fujimoto et al. 2002, Katoh et al. 1994).
Peroxynitrite can oxidise BH4 to the BH3 radical, further reducing BH4 availability to couple eNOS activity and compounding the production of superoxide through uncoupled eNOS activity (Kuzkaya et al. 2003).
In the first of two salvage steps to maintain BH4 levels in the cell, sepiapterin is taken up by the cell and reduced by sepiapterin reductase (SRP) to form BH2 (Sawabe et al. 2008).
Heme iron from the oxygenase domain of eNOS can reduce the BH3 radical back to BH4, with itself being oxidised from the ferrous (Fe2+) back to the ferric (Fe3+) form (Berka et al. 2004).
Mitochondrial COQ3 is an O-methyltransferase required in the reaction to convert 3-demethylubiquinol-10 (DeMQ10H2) to ubiquinol-10 (Q10H2) (Jonassen & Clarke 2000).
A flavin-dependent monooxygenase involved in ubiquinone/ubiquinol biosynthesis, COQ6 (Heeringa et al. 2011) catalyses the C5-hydroxylation of 3-decaprenyl-4-hydroxybenzoic acid (DHB) to 3,4-dihydroxy-5-decaprenylbenzoic acid (DHDB). COQ6 is a peripheral membrane protein that localizes to the matrix side of the inner mitochondrial membrane (Gin et al. 2003). This reaction involving COQ6 is inferred from the equivalent reaction in yeast (Ozeir et al. 2011, Gin et al. 2003).
2-methoxy-6-polyprenyl-1,4-benzoquinol methylase, mitochondrial (COQ5) catalyses the C-methyltransferase conversion of 2-methoxy-6-decaprenyl-1,4-benzoquinol (MDMQ10H2) to 6-methoxy-3-methyl-2-decaprenyl-1,4-benzoquinol (DMQ10H2). This reaction is inferred from the equivalent reaction in yeast (Barkovich et al. 1997).
2-methoxy-6-decaprenylphenol (DMPhOH) is enzymatically converted to 2-methoxy-6-decaprenyl-1,4-benzoquinol (MDMQ10H2). It was thought at one time that the flavin-dependent monooxygenase, COQ6, was the enzyme that catalysed this reaction, however, it has been subsequently shown that COQ6 is not essential for this reaction (Ozeir et al. 2011). However, it is still believed that another member of the COQ family catalyses this event. This reaction is inferred from the equivalent reaction in yeast (Gin et al. 2003, Ozeir et al. 2011).
4-hydroxybenzoate polyprenyltransferase, mitochondrial (COQ2) catalyses the combination of 4-hydroxybenzoic acid, aka para-hydroxybenzoic acid (PHB) with the polyisoprenoid tail all-trans-decaprenyl diphosphate (all-E-10PrP2) to form 3-decaprenyl-4-hydroxybenzoic acid (DHB) (Forsgren et a l. 2004, Tekle et al. 2008). This reaction is irreversible and occurs in the mitochondria.
Mitochondrial COQ3 is an O-methyltransferase required in the reaction to convert 3,4-dihydroxy-5-decaprenylbenzoic acid (DHDB) to 3-methoxy-4-hydroxy-5-decaprenylbenzoic acid (MHDB) (Jonassen & Clarke 2000).
Ubiquinone biosynthesis protein COQ7 homolog (COQ7) (Vajo et al. 1999) catalyses the hydroxylation of 6-methoxy-3-methyl-2-decaprenyl-1,4-benzoquinol (DMQ10H2) to 3-demethylubiquinol-10 (DeMQ10H2). This reaction is inferred from the equivalent reaction in yeast (Marbois & Clarke 1996, Tran et al. 2006). Mitochondrial ubiquinone biosynthesis protein COQ9 is a lipid-binding protein involved in the biosynthesis of coenzyme Q. It binds with COQ7, an interaction that may be necessary to present the lipid to COQ7 activity (Lohman et al. 2014).
3-methoxy-4-hydroxy-5-decaprenylbenzoic acid (MHDB) is enzymatically decarboxylated to form 2-methoxy-6-decaprenylphenol (DMPhOH). At the present time the enzyme identity is unknown but is thought to be a member of the COQ family. This reaction is inferred from the equivalent reaction in yeast (Casey & Threlfall 1978, Goewert et al. 1981).
The polyprenyl diphosphate synthase consists of a tetramer comprising two units of decaprenyl-diphosphate synthase subunit 1 (PDSS1) and two units of decaprenyl-diphosphate synthase subunit 2 (PDSS2). It catalyses the combination of 2-trans,6-trans-farnesyl diphosphate (FPP) with isopentenyl diphosphate (IPPP) to form the polyisoprenoid tail all-trans-decaprenyl diphosphate (all-E-10PrP2) (Saiki et al. 2005, Tekle et al. 2008).
Cytosolic IDH1 (isocitrate dehydrogenase 1) homodimer catalyzes the reaction of isocitrate and NADP+ to form 2-oxoglutarate, CO2, and NADPH + H+. The same enzyme can also localize to peroxisomes (Geisbrecht and Gould 1999; Xu et al. 2004).
Peroxisomal IDH1 (isocitrate dehydrogenase 1) homodimer catalyzes the reaction of isocitrate and NADP+ to form 2-oxoglutarate, CO2, and NADPH + H+. The same enzyme can also localize to the cytosol in at least some cell types (Geisbrecht and Gould 1999; Xu et al. 2004).
A specific transport process that exchanges 2-oxoglutarate for isocitrate across a lipid membrane has been reconstituted in vitro with proteins purified from bovine peroxisomal membranes. The specific protein or proteins that mediate this transport process have not yet been identified in any mammalian system, however (Visser et al. 2006).
In an alternative salvage pathway for BH4 synthesis, dyspropterin (PTHP) may be transformed to sepiapterin, possibly non-enzymatically or by an unknown sepiapterin synthase enzyme (Crabtree & Channon 2011).
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oxide: NOS3 activation and
regulationThe NOS enzymes share a common basic structural organization and requirement for substrate cofactors for enzymatic activity. A central calmodulin-binding motif separates an NH2-terminal oxygenase domain from a COOH-terminal reductase domain. Binding sites for cofactors NADPH, FAD, and FMN are located within the reductase domain, while binding sites for tetrahydrobiopterin (BH4) and heme are located within the oxygenase domain. Once calmodulin binds, it facilitates electron transfer from the cofactors in the reductase domain to heme enabling nitric oxide production. Both nNOS and eNOS contain an additional insert (40-50 amino acids) in the middle of the FMN-binding subdomain that serves as autoinhibitory loop, destabilizing calmodulin binding at low calcium levels and inhibiting electron transfer from FMN to the heme in the absence of calmodulin. iNOS does not contain this insert.
In this Reactome pathway module, details of eNOS activation and regulation are annotated. Originally identified as endothelium-derived relaxing factor, eNOS derived NO is a critical signaling molecule in vascular homeostasis. It regulates blood pressure and vascular tone, and is involved in vascular smooth muscle cell proliferation, platelet aggregation, and leukocyte adhesion. Loss of endothelium derived NO is a key feature of endothelial dysfunction, implicated in the pathogenesis of hypertension and atherosclerosis. The endothelial isoform eNOS is unique among the nitric oxide synthase (NOS) family in that it is co-translationally modified at its amino terminus by myristoylation and is further acylated by palmitoylation (two residues next to the myristoylation site). These modifications target eNOS to the plasma membrane caveolae and lipid rafts.
Factors that stimulate eNOS activation and nitric oxide (NO) production include fluid shear stress generated by blood flow, vascular endothelial growth factor (VEGF), bradykinin, estrogen, insulin, and angiopoietin. The activity of eNOS is further regulated by numerous post-translational modifications, including protein-protein interactions, phosphorylation, and subcellular localization.
Following activation, eNOS shuttles between caveolae and other subcellular compartments such as the noncaveolar plasma membrane portions, Golgi apparatus, and perinuclear structures. This subcellular distribution is variable depending upon cell type and mode of activation.
Subcellular localization of eNOS has a profound effect on its ability to produce NO as the availability of its substrates and cofactors will vary with location. eNOS is primarily particulate, and depending on the cell type, eNOS can be found in several membrane compartments: plasma membrane caveolae, lipid rafts, and intracellular membranes such as the Golgi complex.
Annotated Interactions