Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q8NEX9 (reductase)
 26,410 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Macrophage NO synthase is a homodimer of 130 kDa subunits. Each subunit contains an oxygenase domain that binds iron protoporphyrin IX (heme) and tetrahydrobiopterin (H4biopterin) and a reductase domain that binds FAD, FMN, and calmodulin (CaM) [Ghosh & Stuehr (1995) Biochemistry 34, 801-807]. We have studied the dissociation and unfolding reactions of dimeric iNOS in urea to learn how enzyme structure relates to catalysis and prosthetic group binding. The iNOS dimer dissociated between 0 and 2.5 M urea, and the subunits partially unfolded at 2.5 M urea and above. Dimer dissociation was accompanied by loss of NO synthesis activity and release of bound H4biopterin from the protein. However, the dissociated subunits maintained their cytochrome c and ferricyanide reductase activities and retained near stoichiometric quantities of bound heme. The subunit unfolding transition was accompanied by loss of reductase activities and partial loss of bound heme but retention of bound flavins and CaM. The heme iron in the dissociated subunits remained coordinated through axial cysteine thiolate ligation. Kinetic analysis of dimer dissociation showed that loss of NO synthesis correlated with a loss of heme Soret absorbance at 398 nm and an appearance of absorbance bands at 377 and 460 nm, which were attributed to DTT coordination to the sixth position of the heme iron to form a mixed bisthiolate complex. Subunits could reassociate into a dimer when incubated with L-arginine and H4biopterin. Dimer formation correlated with proportional recoveries of NO synthesis and heme Soret absorbance at 398 nm. Thus, dimeric iNOS undergoes separate dissociation and unfolding transitions in urea, and each transition is accompanied by a loss of a specific catalytic function.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Subunit dissociation and unfolding of macrophage NO synthase: relationship between enzyme structure, prosthetic group binding, and catalytic function. 754 34

Inducible nitric oxide (NO) synthase (iNOS) is comprised of an oxygenase domain containing heme, tetrahydrobiopterin, the substrate binding site, and a reductase domain containing FAD, FMN, calmodulin, and the NADPH binding site. Enzyme activity requires a dimeric interaction between two oxygenase domains with the reductase domains attached as monomeric extensions. To understand how dimerization activates iNOS, we synthesized an iNOS heterodimer comprised of one full-length subunit and one histidine-tagged subunit that was missing its reductase domain. The heterodimer was purified using nickel-Sepharose and 2',5'-ADP affinity chromatography. The heterodimer catalyzed NADPH-dependent NO synthesis from L-arginine at a rate of 52 +/- 6 nmol of NO/min/nmol of heme, which is half the rate of purified iNOS homodimer. Heterodimer NO synthesis was associated with reduction of only half of its heme iron by NADPH, in contrast with near complete heme iron reduction in an iNOS homodimer. Full-length iNOS monomer preparations could not synthesize NO nor catalyze NADPH-dependent heme iron reduction. Thus, dimerization activates NO synthesis by enabling electrons to transfer between the reductase and oxygenase domains. Although a single reductase domain can reduce only one of two hemes in a dimer, this supports NO synthesis from L-arginine.
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PMID:Heme iron reduction and catalysis by a nitric oxide synthase heterodimer containing one reductase and two oxygenase domains. 863 49

Nitric oxide synthase (EC 1.14.13.39) binds arginine and NADPH as substrates, and FAD, FMN, tetrahydrobiopterin, haem and calmodulin as cofactors. The protein consists of a central calmodulin-binding sequence flanked on the N-terminal side by a haem-binding region, analogous to cytochrome P-450, and on the C-terminal side by a region homologous with NADPH:cytochrome P-450 reductase. The structure of recombinant rat brain nitric oxide synthase was analysed by limited proteolyis. The products were identified by using antibodies to defined sequences, and by N-terminal sequencing. Low concentrations of trypsin produced three fragments, similar to those in a previous report [Sheta, McMillan and Masters (1994) J. Biol. Chem. 269, 15147-15153]: that of Mr approx. 135000 (N-terminus Gly-221) resulted from loss of the N-terminal extension (residues 1-220) unique to neuronal nitric oxide synthase. The fragments of Mr 90000 (haem region) and 80000 (reductase region, N-terminus Ala-728) were produced by cleavage within the calmodulin-binding region. With more extensive trypsin treatment, these species were shown to be transient, and three smaller, highly stable fragments of Mr 14000 (N-terminus Leu-744 within the calmodulin region), 60000 (N-terminus Gly-221) and 63000 (N-terminus Lys-856 within the FMN domain) were formed. The species of Mr approx. 60000 represents a domain retaining haem and nitroarginine binding. The two species of Mr 63000 and 14000 remain associated as a complex. This complex retains cytochrome c reductase activity, and thus is the complete reductase region, yet cleaved at Lys-856. This cleavage occurs within a sequence insertion relative to the FMN domain present in inducible nitric oxide synthase. Prolonged proteolysis treatment led to the production of a protein of Mr approx. 53000 (N-terminus Ala-953), corresponding to a cleavage between the FMN and FAD domains. The major products after chymotryptic digestion were similar to those with trypsin, although the pathway of intermediates differed. The haem domain was smaller, starting at residue 275, yet still retained the arginine binding site. These data have allowed us to identify stable domains representing both the arginine/haem-binding and the reductase regions.
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PMID:Identification of the domains of neuronal nitric oxide synthase by limited proteolysis. 866 Mar 10

Nitric oxide synthase (EC 1.14.13.39) is a homodimer. Limited proteolysis has previously shown that it consists of two major domains. The C-terminal or reductase domain binds FMN, FAD and NADPH. The N-terminal or oxygenase domain is known to bind arginine, (6R)-5,6,7,8-tetrahydro-l-biopterin (tetrahydrobiopterin) and haem. The exact residues of the inducible nitric oxide synthase (iNOS) protein involved in binding to these molecules have yet to be identified, although the haem moiety is known to be co-ordinated through a cysteine thiolate ligand. We have expressed two forms of the haem-binding domain of human iNOS (residues 1-504 and 59-504) in Escherichia coli as glutathione S-transferase (GST) fusion proteins. The iNOS 1-504 and 59-504 fusion proteins bound similar amounts of haem, Nomega-nitro-l-arginine (nitroarginine) and tetrahydrobiopterin, showing that the first 58 residues are not required for binding these factors. Using site-directed mutagenesis we have mutated Cys-200, Cys-217, Cys-228, Cys-290, Cys-384 and Cys-457 to alanine residues within the iNOS 59-504 haem-binding domain. Mutation of Cys-200 resulted in a complete loss of haem, nitroarginine and tetrahydrobiopterin binding. Mutants of Cys-217, Cys-228, Cys-290, Cys-384 or Cys-457 showed no effect on the haem content of the fusion protein, no effect on the reduced CO spectral peak (444 nm) and were able to bind nitroarginine and tetrahydrobiopterin at levels equivalent to the wild-type fusion protein. After removal of the GST polypeptide, the wild-type iNOS 59-504 domain was dimeric, whereas the C200A mutant form was monomeric. When the mutated domains were incorporated into a reconstructed full-length iNOS protein expressed in Xenopus oocytes, only the Cys-200 mutant showed a loss of catalytic activity: all the other mutant iNOS proteins showed near wild-type enzymic activity. From this systematic approach we conclude that although Cys-217, Cys-228, Cys-290, Cys-384 and Cys-457 are conserved in all three NOS isoforms they are not essential for cofactor or substrate binding or for enzymic activity of iNOS, and that Cys-200 provides the proximal thiolate ligand for haem binding in human iNOS.
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PMID:Cysteine-200 of human inducible nitric oxide synthase is essential for dimerization of haem domains and for binding of haem, nitroarginine and tetrahydrobiopterin. 917 73

Inducible nitric oxide synthase (iNOS or NOSII) is one of three distinct NOS isoforms in human. The NOSII isoform is expressed in a variety of cells and tissues in response to endotoxins and cytokines. The human genome contains at least two loci for the NOSII gene, one of which (NOSII-1) has previously been assigned to proximal region of the long arm (cen-q11.2 or q11.2-q12) or to pericentric (p11-q11) regions of chromosome 17. The present study, carried out using fluorescence in situ hybridization (FISH) method, shows that a pseudogene gene (NOSII-2) is mapped to chromosome 17q11.2 site. The NOSII-2 sequence contains the exon and intron sequences present in NOSII-1 but with several mutations such as single base substitutions, additions, and deletions. Additionally, the NOSII-2 sequence also contains an incomplete reductase domain which corresponds only to the cofactor binding sites without the oxygenase domain that carry heme and substrate binding sites. NOSII-2, therefore, appears to be an unprocessed pseudogene, which cannot be translated to a functional enzyme because of its incomplete sequences and mutations.
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PMID:An unprocessed pseudogene of inducible nitric oxide synthase gene in human. 944 1

NO synthases are unique among eukaryotic enzymes in being dimeric, calmodulin-dependent or calmodulin-containing cytochrome P-450-like haemoproteins that combine reductase and oxygenase catalytic domains in one monomer. They catalyse the formation of NO from L-arginine in the presence of NADPH and molecular oxygen. There are, broadly, three distinctive forms of NO synthase, of which two are constitutively expressed in a variety of cells and are calcium dependent. Of these, the endothelial cell-specific form (eNOS) can play an important role in vascular development, maintenance of vascular tone and tumour growth. A third, inducible, calcium-independent form (iNOS), is important in the immunogenic and cytotoxic response of T-lymphocytes and macrophages. NO acts as an intracellular secondary messenger and provides an efficient system for cellular regulation, interaction and defence, while striking a very fine balance in its role in tumour growth and--under some circumstances--appearing to promote tumour growth, whereas other evidence suggests its production can be growth inhibitory. Nevertheless, tumour cells do express both the constitutive and inducible forms of NO synthase, albeit at widely different levels, and their presence in some human cancers correlates positively with tumour grade. Its role is strictly dependent upon its chemical reactivity with oxygen and metals, e.g. in haem-containing proteins, rather than specific structural interactions with physiological targets. Conflicting evidence still surrounds the effects of expressing high levels of iNOS activity and consequent production of NO on tumour growth. Similar conflicting results have been obtained by applying various NO donors and NO synthase inhibitors. Overall, NO may be acting as part of a signalling cascade for neovascularization in vivo, whereas in vitro cytotoxic properties contribute to the 'apparent' slowing of the growth of cells. It is our contention that low concentrations of NO can be pro-angiogenic and pro-tumour growth, whereas higher NO concentrations can have the opposite effect. Like many other areas of therapeutics, the concept of dose-response is very important. Modification of NO synthase activity in tumours, and hence NO biosynthesis, may be regarded as a promising means for selective tumour blood flow modification and provides a novel approach for reducing tumour oxygenation aimed at enhancing the efficiency of hypoxia-mediated, bioreductively activated anti-cancer drugs.
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PMID:Role of nitric oxide in growth of solid tumours: a balancing act. 949 11

Crystal structures of the murine cytokine-inducible nitric oxide synthase oxygenase dimer with active-center water molecules, the substrate L-arginine (L-Arg), or product analog thiocitrulline reveal how dimerization, cofactor tetrahydrobiopterin, and L-Arg binding complete the catalytic center for synthesis of the essential biological signal and cytotoxin nitric oxide. Pterin binding refolds the central interface region, recruits new structural elements, creates a 30 angstrom deep active-center channel, and causes a 35 degrees helical tilt to expose a heme edge and the adjacent residue tryptophan-366 for likely reductase domain interactions and caveolin inhibition. Heme propionate interactions with pterin and L-Arg suggest that pterin has electronic influences on heme-bound oxygen. L-Arginine binds to glutamic acid-371 and stacks with heme in an otherwise hydrophobic pocket to aid activation of heme-bound oxygen by direct proton donation and thereby differentiate the two chemical steps of nitric oxide synthesis.
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PMID:Structure of nitric oxide synthase oxygenase dimer with pterin and substrate. 951 16

The inducible nitric oxide synthase (iNOS) contains an amino-terminal oxygenase domain, a carboxy-terminal reductase domain, and an intervening calmodulin-binding region. For the synthesis of nitric oxide (NO), iNOS is active as a homodimer. The human iNOS mRNA is subject to alternative splicing, including deletion of exons 8 and 9 that encode amino acids 242-335 of the oxygenase domain. In this study, iNOS8(-)9(-) and full-length iNOS (iNOSFL) were cloned from bronchial epithelial cells. Expression of iNOS8(-)9(-) in 293 cell line resulted in generation of iNOS8(-)9(-) mRNA and protein but did not lead to NO production. In contrast to iNOSFL, iNOS8(-)9(-) did not form dimers. Similar to iNOSFL, iNOS8(-)9(-) exhibited NADPH-diaphorase activity and contained tightly bound calmodulin, indicating that the reductase and calmodulin-binding domains were functional. To identify sequences in exons 8 and 9 that are critical for dimerization, iNOSFL was used to construct 12 mutants, each with deletion of eight residues in the region encoded by exons 8 and 9. In addition, two "control" iNOS deletion mutants were synthesized, lacking either residues 45-52 of the oxygenase domain or residues 1131-1138 of the reductase domain. Whereas both control deletion mutants generated NO and formed dimers, none of the 12 other mutants formed dimers or generated NO. The region encoded by exons 8 and 9 is critical for iNOS dimer formation and NO production but not for reductase activity. This region could be a potential target for therapeutic interventions aimed at inhibiting iNOS dimerization and hence NO synthesis.
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PMID:Cloning and characterization of human inducible nitric oxide synthase splice variants: a domain, encoded by exons 8 and 9, is critical for dimerization. 963

Cytokine-inducible nitric-oxide (NO) synthase (iNOS) contains an oxygenase domain that binds heme, tetrahydrobiopterin, and L-arginine, and a reductase domain that binds FAD, FMN, calmodulin, and NADPH. Dimerization of two oxygenase domains allows electrons to transfer from the flavins to the heme irons, which enables O2 binding and NO synthesis from L-arginine. In an iNOS heterodimer comprised of one full-length subunit and an oxygenase domain partner, the single reductase domain transfers electrons to only one of two hemes (Siddhanta, U., Wu, C., Abu-Soud, H. M., Zhang, J., Ghosh, D. K., and Stuehr, D. J. (1996) J. Biol. Chem. 271, 7309-7312). Here, we characterize a pair of heterodimers that contain an L-Arg binding mutation (E371A) in either the full-length or oxygenase domain subunit to identify which heme iron becomes reduced. The E371A mutation prevented L-Arg binding to one oxygenase domain in each heterodimer but did not affect the L-Arg affinity of its oxygenase domain partner and did not prevent heme iron reduction in any case. The mutation prevented NO synthesis when it was located in the oxygenase domain of the adjacent subunit but had no effect when in the oxygenase domain in the same subunit as the reductase domain. Resonance Raman characterization of the heme-L-Arg interaction confirmed that E371A only prevents L-Arg binding in the mutated oxygenase domain. Thus, flavin-to-heme electron transfer proceeds exclusively between adjacent subunits in the heterodimer. This implies that domain swapping occurs in an iNOS dimer to properly align reductase and oxygenase domains for NO synthesis.
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PMID:Domain swapping in inducible nitric-oxide synthase. Electron transfer occurs between flavin and heme groups located on adjacent subunits in the dimer. 966 73

Nitric oxide (NO) is produced by nitric oxide synthases (cNOS and iNOS) in endothelial cells upon stimulation by various agents like Ca(2+)-calmodulin, cytokines and TNF. It acts as a paracrine on adjacent cells to activate soluble guanylyl cyclase in the production of cGMP, a second messenger in signal transduction cascades, leading to various cellular responses. The circulating blood contains certain steady-state concentrations of NO in the plasma in order to maintain normal vascular tone and other appropriate conditions for the systemic and pulmonary circulation. This homeostasis of NO in the rapidly moving blood must be maintained by a delicate balance between its production by NOSs and its instant scavenging by hemoglobin (Hb) in the erythrocytes. Under physiological conditions ([NO] <<< [Hb]), NO is sequestered by deoxy Hb to form alpha-nitrosyl Hb, alpha (Fe-NO)2 beta (Fe)2, where NO is tightly (KD = 10(-12) M) bound to the alpha-subunits. Upon binding NO to the alpha-subunbits, Hb shifts its conformation to a T-(low-affinity extreme) state and its beta-subunits become an efficient O2 carrier. The same molecular mechanism of NO-induced conformation change operates in both Hb and soluble guanylyl cyclase. This is caused by the NO-induced trans-axial cleavage of the heme Fe-proximal His bonds in these hemoproteins. This bond cleavage mechanism allows Hb to survive as an effective O2 carrier even after sequestration of NO. The NO sequestered in Hb is eventually oxidized aerobically to NO3- in the reaction of Fe-NO + O2-->Fe(+) + NO3. Met Hb (Fe+) so formed is cycled back to deoxy Hb (Fe) by intra-erythrocyte Hb reductase to complete the NO scavenging. Thus, the NO in the blood acts on soluble guanylyl cyclase in vascular smooth muscles to dilate the blood vessels to increase blood delivery, whereas excess NO in the blood, which is sequestered by Hb, could help Hb to deliver O2 more efficiently in peripheral tissues.
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PMID:[Nitric oxide and hemoglobin]. 979 69


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