Ikuko Sagami

The 42-Amino Acid Insert in the FMN Domain of Neuronal Nitric-oxide Synthase Exerts Control over Ca2+/Calmodulin-dependent Electron Transfer

The neuronal and endothelial nitric-oxide synthases (nNOS and eNOS) differ from inducible NOS in their dependence on the intracellular Ca2+ concentration. Both nNOS and eNOS are activated by the reversible binding of calmodulin (CaM) in the presence of Ca2+, whereas inducible NOS binds CaM irreversibly. One major divergence in the close sequence similarity between the NOS isoforms is a 40–50-amino acid insert in the middle of the FMN-binding domains of nNOS and eNOS. It has previously been proposed that this insert forms an autoinhibitory domain designed to destabilize CaM binding and increase its Ca2+ dependence. To examine the importance of the insert we constructed two deletion mutants designed to remove the bulk of it from nNOS. Both mutants (Δ40 and Δ42) retained maximal NO synthesis activity at lower concentrations of free Ca2+ than the wild type enzyme. They were also found to retain 30% of their activity in the absence of Ca2+/CaM, indicating that the insert plays an important role in disabling the enzyme when the physiological Ca2+concentration is low. Reduction of nNOS heme by NADPH under rigorous anaerobic conditions was found to occur in the wild type enzyme only in the presence of Ca2+/CaM. However, reduction of heme in the Δ40 mutant occurred spontaneously on addition of NADPH in the absence of Ca2+/CaM. This suggests that the insert regulates activity by inhibiting electron transfer from FMN to heme in the absence of Ca2+/CaM and by destabilizing CaM binding at low Ca2+ concentrations, consistent with its role as an autoinhibitory domain.

Restriction fragment length polymorphism of the human CYP2E1 (cytochrome P450IIE1) gene and susceptibility to lung cancer: possible relevance to low smoking exposure.

Polymorphic metabolism of certain chemical carcinogens may result in differences in susceptibility to cancers. Human CYP2E1 (cytochrome P450IIE1) is an enzyme involved in the metabolic activation of precarcinogens such as nitrosamines. We detected a restriction fragment length polymorphism (RFLP) of the human CYP2E1 gene for the restriction endonuclease Dra I. The distribution of this polymorphism was examined among lung cancer patients (n = 91), patients with cancer of the digestive tract (n = 45) and controls (n = 76). A significant difference in the distribution was observed between lung cancer patients and controls (chi 2 = 11.4 with 2 df; p < 0.005). On the other hand, there was no significant difference between patients between cancer of the digestive tract and controls (chi 2 = 4.87 with 2 df; NS). This finding suggests that the Dra I polymorphism of the CYP2E1 gene is associated with susceptibility to lung cancer. In addition, an association was found between the amount of lifelong smoking exposure and the distribution of the genotypes of the RFLP among lung cancer patients. The distribution pattern seemed deviated from that of controls especially in the population of low smoking exposure. Our Northern blot analysis data using RNA from human liver autopsy samples suggest that the Dra I polymorphism might be associated with the gene expression of CYP2E1 at mRNA level.

Characterization of a direct oxygen sensor heme protein from Escherichia coli. Effects of the heme redox states and mutations at the heme-binding site on catalysis and structure.

A protein containing a heme-binding PAS (PAS is from the protein names in which imperfect repeat sequences were first recognized: PER, ARNT, andSIM) domain from Escherichia coli has been implied a direct oxygen sensor (Ec DOS) enzyme. In the present study, we isolated cDNA for the Ec DOS full-length protein, expressed it in E. coli, and examined its structure-function relationships for the first time. EcDOS was found to be tetrameric and was obtained as a 6-coordinate low spin ferric heme complex. Its α-helix content was calculated as 53% by CD spectroscopy. The redox potential of the heme was found to be +67 mV versus SHE. Mutation of His-77 of the isolated PAS domain abolished heme binding, whereas mutation of His-83 did not, suggesting that His-77 is one of the heme axial ligands. Ferrous, but not ferric, Ec DOS had phosphodiesterase (PDE) activity of nearly 0.15 min−1 with cAMP, which was optimal at pH 8.5 in the presence of Mg2+ and was strongly inhibited by CO, NO, and etazolate, a selective cAMP PDE inhibitor. Absorption spectral changes indicated tight CO and NO bindings to the ferrous heme. Therefore, the present study unequivocally indicates for the first time that Ec DOS exhibits PDE activity with cAMP and that this is regulated by the heme redox state.

Identification of Caveolin-1-interacting Sites in Neuronal Nitric-oxide Synthase MOLECULAR MECHANISM FOR INHIBITION OF NO FORMATION

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 IC50 value of 1.8 μm. Mutations of Phe584 and Trp587 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 (Δ40) and a C-terminal truncation mutant (ΔC33), 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 CaM-independent, NO formation activities of both Δ40 and ΔC33, suggesting that CaV1p1 inhibits interdomain electron transfer induced by CaM from the reductase domain to the oxygenase domain.

CO-dependent activity-controlling mechanism of heme-containing CO-sensor protein, neuronal PAS domain protein 2.

Neuronal PAS domain protein 2, which was recently established to be a heme protein, acts as a CO-dependent transcription factor. The protein consists of the basic helix-loop-helix domain and two heme-containing PAS domains (PAS-A and PAS-B). In this study, we prepared wild type and mutants of the isolated PAS-A domain and measured resonance Raman spectra of these proteins. Upon excitation of the Raman spectrum at 363.8 nm, a band assignable to Fe3+-S stretching was observed at 334 cm–1 for the ferric wild type protein; in contrast, this band was drastically weaker in the spectrum of C170A, suggesting that Cys170 is an axial ligand of the ferric heme. The Raman spectrum of the reduced form of wild type was mainly of six-coordinate low spin, and the ν11 band, which is sensitive to the donor strength of the axial ligand, was lower than that of reduced cytochrome c3, suggesting coordination of a strong ligand and thus a deprotonated His. In the reduced forms of H119A and H171A, the five-coordinate species became more prevalent, whereas no such changes were observed for C170A, indicating that His119 and His171, but not Cys170, are axial ligands in the ferrous heme. This means that ligand replacement from Cys to His occurs upon heme reduction. The νFe-CO versus νC-O correlation indicates that a neutral His is a trans ligand of CO. Our results support a mechanism in which CO binding disrupts the hydrogen bonding of His171 with surrounding amino acids, which induces conformational changes in the His171-Cys170 moiety, leading to physiological signaling.

Human Geranylgeranyl Diphosphate Synthase cDNA CLONING AND EXPRESSION

Geranylgeranyl diphosphate (GGPP) synthase (GGPPSase) catalyzes the synthesis of GGPP, which is an important molecule responsible for the C20-prenylated protein biosynthesis and for the regulation of a nuclear hormone receptor (LXR·RXR). The human GGPPSase cDNA encodes a protein of 300 amino acids which shows 16% sequence identity with the known human farnesyl diphosphate (FPP) synthase (FPPSase). The GGPPSase expressed inEscherichia coli catalyzes the GGPP formation (240 nmol/min/mg) from FPP and isopentenyl diphosphate. The human GGPPSase behaves as an oligomeric molecule with 280 kDa on a gel filtration column and cross-reacts with an antibody directed against bovine brain GGPPSase, which differs immunochemically from bovine brain FPPSase. Northern blot analysis indicates the presence of two forms of the mRNA.

Crucial Role of Lys423 in the Electron Transfer of Neuronal Nitric-oxide Synthase

Nitric-oxide synthase (NOS) is composed of an oxygenase domain having cytochrome P450-type heme active site and a reductase domain having FAD- and FMN-binding sites. To investigate the route of electron transfer from the reductase domain to the heme, we generated mutants at Lys423 in the heme proximal site of neuronal NOS and examined the catalytic activities, electron transfer rates, and NADPH oxidation rates. A K423E mutant showed no NO formation activity (<0.1 nmol/min/nmol heme), in contrast with that (72 nmol/min/nmol heme) of the wild type enzyme. The electron transfer rate (0.01 min−1) of the K423E on addition of excess NADPH was much slower than that (>10 min−1) of the wild type enzyme. From the crystal structure of the oxygenase domain of endothelial NOS, Lys423 of neuronal NOS is likely to interact with Trp409 which lies in contact with the heme plane and with Cys415, the axial ligand. It is also exposed to solvent and lies in the region where the heme is closest to the protein surface. Thus, it seems likely that ionic interactions between Lys423 and the reductase domain may help to form a flavin to heme electron transfer pathway.

The Crucial Roles of Asp-314 and Thr-315 in the Catalytic Activation of Molecular Oxygen by Neuronal Nitric-oxide Synthase A SITE-DIRECTED MUTAGENESIS STUDY

Nitric-oxide synthase (NOS) is a flavohemoprotein that has a cytochrome P450 (P450)-type heme active site and catalyzes the monooxygenation of l-Arg toN G-hydroxy-l-Arg (NHA) according to the normal P450-type reaction in the first step of NO synthesis. However, there is some controversy as to how the second step of the reaction, from NHA to NO and l-citrulline, occurs within the P450 domain of NOS. By referring to the heme active site of P450, it is conjectured that polar amino acid(s) such as Asp/Glu and Thr must be responsible for the activation of molecular oxygen in NOS. In this study, we have created Asp-314 → Ala and Thr-315 → Ala mutants of neuronal NOS, both of which had absorption maxima at 450 nm in the spectra of the CO-reduced complexes and studied NO formation rates and other kinetic parameters as well as the substrate binding affinity. The Asp-314 → Ala mutant totally abolished NO formation activity and markedly increased the rate of H2O2 formation by 20-fold compared with the wild type when l-Arg was used as the substrate. The NADPH oxidation and O2 consumption rates for the Asp-314 → Ala mutant were 60–65% smaller than for the wild type. The Thr-315 → Ala mutant, on the other hand, retained NO formation activity that was 23% higher than the wild type, but like the Asp-314 → Ala mutation, markedly increased the H2O2 formation rate. The NADPH oxidation and O2 consumption rates for the Thr-315 → Ala mutant were, respectively, 56 and 27% higher than for the wild type. When NHA was used as the substrate, similar values were obtained. Thus, we propose that Asp-314 is crucial for catalysis, perhaps through involvement in the stabilization of an oxygen-bound intermediate. An important role for Thr-315 in the catalysis is also suggested.

Spectroscopic and DNA-binding characterization of the isolated heme-bound basic helix–loop–helix-PAS-A domain of neuronal PAS protein 2 (NPAS2), a transcription activator protein associated with circadian rhythms

Neuronal PAS domain protein 2 (NPAS2) is a circadian rhythm‐associated transcription factor with two heme‐binding sites on two PAS domains. In the present study, we compared the optical absorption spectra, resonance Raman spectra, heme‐binding kinetics and DNA‐binding characteristics of the isolated fragment containing the N‐terminal basic helix–loop–helix (bHLH) of the first PAS (PAS‐A) domain of NPAS2 with those of the PAS‐A domain alone. We found that the heme‐bound bHLH‐PAS‐A domain mainly exists as a dimer in solution. The Soret absorption peak of the Fe(III) complex for bHLH‐PAS‐A (421 nm) was located at a wavelength 9 nm higher than for isolated PAS‐A (412 nm). The axial ligand trans to CO in bHLH‐PAS‐A appears to be His, based on the resonance Raman spectra. In addition, the rate constant for heme association with apo‐bHLH‐PAS (3.3 × 107 mol−1·s−1) was more than two orders of magnitude higher than for association with apo‐PAS‐A (< 105 mol−1·s−1). These results suggest that the bHLH domain assists in stable heme binding to NPAS2. Both optical and resonance Raman spectra indicated that the Fe(II)–NO heme complex is five‐coordinated. Using the quartz‐crystal microbalance method, we found that the bHLH‐PAS‐A domain binds specifically to the E‐box DNA sequence in the presence, but not in the absence, of heme. On the basis of these results, we discuss the mode of heme binding by bHLH‐PAS‐A and its potential role in regulating DNA binding.

Interactions between the isolated oxygenase and reductase domains of neuronal nitric-oxide synthase: Assessing the role of calmodulin

Nitric-oxide synthase (NOS) is a fusion protein composed of an oxygenase domain with a heme-active site and a reductase domain with an NADPH binding site and requires Ca2+/calmodulin (CaM) for NO formation activity. We studied NO formation activity in reconstituted systems consisting of the isolated oxygenase and reductase domains of neuronal NOS with and without the CaM binding site. Reductase domains with 33-amino acid C-terminal truncations were also examined. These were shown to have faster cytochrome c reduction rates in the absence of CaM.NG -hydroxy-l-Arg, an intermediate in the physiological NO synthesis reaction, was found to be a viable substrate. Turnover rates forNG -hydroxy-l-Arg in the absence of Ca2+/CaM in most of the reconstituted systems were 2.3–3.1 min−1. Surprisingly, the NO formation activities with CaM binding sites on either reductase or oxygenase domains were decreased dramatically on addition of Ca2+/CaM. However, NADPH oxidation and cytochrome c reduction rates were increased by the same procedure. Activation of the reductase domains by CaM addition or by C-terminal deletion failed to increase the rate of NO synthesis. Therefore, both mechanisms appear to be less important than the domain-domain interaction, which is controlled by CaM binding in wild-type neuronal NOS, but not in the reconstituted systems.