Crystallographic Studies on Endothelial Nitric Oxide Synthase Complexed with Nitric Oxide and Mechanism-Based Inhibitors

Li, Huiying; Raman, C.S.; Martásek, Pavel; Masters, B.S.S.; Poulos, T.L.

doi:10.1021/bi002658v

Cited by 76 publications

(99 citation statements)

References 48 publications

(87 reference statements)

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“…Based on the structure of eNOSoxy complexed with NIO, Poulos and co-workers (17) favored the first heme oxygenase-like mechanism since the methyl carbon of NIO is closest to the B nitrogen of the porphyrin but ϳ4 Å away from the ␣-meso carbon. They suggested that this inactivation mechanism also applies to W1400 and explained the observation that W1400 is an irreversible activator only for iNOS by its higher turnover number compared with eNOS and nNOS (17). Although this may be an important kinetic factor, our structures suggest additional reasons.…”

Section: Figmentioning

confidence: 56%

“…The latter group is held in place by interactions with the hydroxyl groups of Tyr-485/706 (iNOSoxy/nNOSoxy) and the cofactor H 4 B, respectively. The absence of H 4 B in the structure of the eNOS W1400 complex (17) might be the reason why there is only some residual electron density for the 3-aminomethyl group (17). Based on the available data, it …”

Section: Structures Of the N -Propyl-l-arg Complexes-npa Belongs To Tmentioning

confidence: 99%

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Structural Basis for the Specificity of the Nitric-oxide Synthase Inhibitors W1400 and N ω-Propyl-l-Arg for the Inducible and Neuronal Isoforms

Fedorov

Hartmann

Ghosh

et al. 2003

Journal of Biological Chemistry

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The high level of amino acid conservation and structural similarity in the immediate vicinity of the substrate binding sites of the oxygenase domains of the nitric-oxide synthase (NOS) isoforms (eNOSoxy, iNOSoxy, and nNOSoxy) make the interpretation of the structural basis of inhibitor isoform specificity a challenge and provide few clues for the design of new selective compounds. Crystal structures of iNOSoxy and nNOSoxy complexed with the inhibitors W1400 and N -propyl-Larginine provide a rationale for their isoform specificity. It involves differences outside the immediate active site as well as a conformational flexibility in the active site that allows the adoption of distinct conformations in response to interactions with the inhibitors. This flexibility is determined by isoform-specific residues outside the active site.

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Section: Figmentioning

confidence: 56%

Section: Structures Of the N -Propyl-l-arg Complexes-npa Belongs To Tmentioning

confidence: 99%

Structural Basis for the Specificity of the Nitric-oxide Synthase Inhibitors W1400 and N ω-Propyl-l-Arg for the Inducible and Neuronal Isoforms

Fedorov

Hartmann

Ghosh

et al. 2003

Journal of Biological Chemistry

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“…The cDNA sequence encoding full-length caveolin-3 (residues 1-151) was cloned by PCR using the rat skeletal muscle cDNA library (Stratagene, La Jolla, CA) and primers containing BamHI and EcoRI restriction sites for subcloning at the 5Ј-and 3Ј-ends, respectively. Each construct was confirmed by direct sequencing, and the fusion proteins were subsequently expressed in E. coli BL21 (DE3) cells and purified by affinity chromatography using Ni 2ϩ -NTA-agarose, as described by Li et al (37). The GST-caveolin-1 fusion protein was constructed by subcloning the full-length caveolin cDNA into the pGEX4-T-2 expression vector.…”

Section: Methodsmentioning

confidence: 99%

Identification of Caveolin-1-interacting Sites in Neuronal Nitric-oxide Synthase

Sato

Sagami

Shimizu

2004

Journal of Biological Chemistry

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Caveolin is known to down-regulate both neuronal (nNOS) and endothelial nitric-oxide synthase (eNOS). In the present study, direct interactions of recombinant caveolin-1 with both the oxygenase and reductase domains of nNOS were demonstrated using in vitro binding assays. To elucidate the mechanism of nNOS regulation by caveolin, we examined the effects of a caveolin-1 scaffolding domain peptide (CaV1p1; residues (82-101)) on the catalytic activities of wild-type and mutant nNOSs. CaV1p1 inhibited NO formation activity and NADPH oxidation of wild-type nNOS in a dose-dependent manner with an IC 50 value of 1.8 M. Mutations of Phe 584 and Trp 587 within a caveolin binding consensus motif of the oxygenase domain did not result in the loss of CaV1p1 inhibition, indicating that an alternate region of nNOS mediates inhibition by caveolin. The addition of CaV1p1 also inhibited more than 90% of the cytochrome c reductase activity in the isolated reductase domain with or without the calmodulin (CaM) binding site, whereas CaV1p1 inhibited ferricyanide reductase activity by only 50%. These results suggest that there are significant differences in the mechanism of inhibition by caveolin for nNOS as compared with those previously reported for eNOS. Further analysis of the interaction through the use of several reductase domain deletion mutants revealed that the FMN domain was essential for successful interaction between caveolin-1 and nNOS reductase. We also examined the effects of CaV1p1 on an autoinhibitory domain deletion mutant (⌬40) and a C-terminal truncation mutant (⌬C33), both of which are able to form NO in the absence of CaM, unlike the wild-type enzyme. Interestingly, CaV1p1 inhibited CaM-dependent, but not CaMindependent, NO formation activities of both ⌬40 and ⌬C33, suggesting that CaV1p1 inhibits interdomain electron transfer induced by CaM from the reductase domain to the oxygenase domain.Nitric-oxide synthase (NOS) 1 generates the physiologically important nitric oxide molecule (NO) from L-arginine (L-Arg) (1-5).NOS consists of two functional domains: an N-terminal oxygenase domain containing a cytochrome P450 (P450)-like heme active site, and L-Arg and (6R)-5,6,7,8-tetrahydrobiopterin (H 4 B) binding sites (6), and a C-terminal reductase domain that contains the FAD, FMN, and NADPH binding sites (7,8). The two domains are connected by a calmodulin (CaM) binding site. Binding of CaM facilitates the interdomain electron transfers required for NO formation as well as intradomain electron transfers within the reductase domain. Two isoforms of NOS (neuronal NOS (nNOS) and endothelial NOS (eNOS)) are constitutively expressed and are regulated by intracellular Ca 2ϩ concentrations via the reversible binding of Ca 2ϩ /CaM (9). In contrast, the inducible NOS isoform (iNOS) is regulated at the transcriptional level, and binds CaM tightly even at basal Ca 2ϩ concentrations (10). CaM-dependent regulation of the three NOS isoforms is also controlled by isoform-specific sequences (i.e. insertions or extensions) and by pho...

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“…Certain fungal CPOs and bacterial P450s have been genetically engineered for large-scale biotransformations (7)(8)(9)(10). The active sites of these three protein families, known in detail from a number of x-ray crystal structures (4,(11)(12)(13), are remarkably similar. All three have an iron-protoporphyrin IX center coordinated to a cysteine thiolate.…”

Section: Oxygen Activation By Heme Proteinsmentioning

confidence: 99%

The bioinorganic chemistry of iron in oxygenases and supramolecular assemblies

Groves

2003

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

305

174

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The bioinorganic chemistry of iron is central to life processes. Organisms must recruit iron from their environment, control iron storage and trafficking within cells, assemble the complex, iron-containing redox cofactors of metalloproteins, and manage a myriad of biochemical transformations by those enzymes. The coordination chemistry and the variable oxidation states of iron provide the essential mechanistic machinery of this metabolism. Our current understanding of several aspects of the chemistry of iron in biology are discussed with an emphasis on the oxygen activation and transfer reactions mediated by heme and nonheme iron proteins and the interactions of amphiphilic iron siderophores with lipid membranes.

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Crystallographic Studies on Endothelial Nitric Oxide Synthase Complexed with Nitric Oxide and Mechanism-Based Inhibitors

Cited by 76 publications

References 48 publications

Structural Basis for the Specificity of the Nitric-oxide Synthase Inhibitors W1400 and N ω-Propyl-l-Arg for the Inducible and Neuronal Isoforms

Structural Basis for the Specificity of the Nitric-oxide Synthase Inhibitors W1400 and N ω-Propyl-l-Arg for the Inducible and Neuronal Isoforms

Identification of Caveolin-1-interacting Sites in Neuronal Nitric-oxide Synthase

The bioinorganic chemistry of iron in oxygenases and supramolecular assemblies

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