The emerging roles of nitric oxide (NO) in plant mitochondria

In recent years nitric oxide (NO) has been recognized as an important signal molecule in plants. Both, reductive and oxidative pathways and different subcellular compartments appear involved in NO production. The reductive pathway uses nitrite as substrate, which is exclusively generated by cytosolic nitrate reductase (NR) and can be converted to NO by the same enzyme. The mitochondrial electron transport chain is another site for nitrite to NO reduction, operating specifically when the normal electron acceptor, O₂, is low or absent. Under these conditions, the mitochondrial NO production contributes to hypoxic survival by maintaining a minimal ATP formation. In contrast, excessive NO production and concomitant nitrosative stress may be prevented by the operation of NO-scavenging mechanisms in mitochondria and cytosol. During pathogen attacks, mitochondrial NO serves as a nitrosylating agent promoting cell death; whereas in symbiotic interactions as in root nodules, the turnover of mitochondrial NO helps in improving the energy status similarly as under hypoxia/anoxia. The contribution of NO turnover during pathogen defense, symbiosis and hypoxic stress is discussed in detail.     

Structural Basis for Catalysis by the Mono- and Dimetalated Forms of the dapE-Encoded N-succinyl-l,l-Diaminopimelic Acid Desuccinylase

Biosynthesis of lysine and meso-diaminopimelic acid in bacteria provides essential components for protein synthesis and construction of the bacterial peptidoglycan cell wall. The dapE operon enzymes synthesize both meso-diaminopimelic acid and lysine and, therefore, represent potential targets for novel antibacterials. The dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase functions in a late step of the pathway and converts N-succinyl-l,l-diaminopimelic acid to l,l-diaminopimelic acid and succinate. Deletion of the dapE gene is lethal to Helicobacter pylori and Mycobacterium smegmatis, indicating that DapE's are essential for cell growth and proliferation. Since there are no similar pathways in humans, inhibitors that target DapE may have selective toxicity against only bacteria. A major limitation in developing antimicrobial agents that target DapE has been the lack of structural information. Herein, we report the high-resolution X-ray crystal structures of the DapE from Haemophilus influenzae with one and two zinc ions bound in the active site, respectively. These two forms show different activity. Based on these newly determined structures, we propose a revised catalytic mechanism of peptide bond cleavage by DapE enzymes. These structures provide important insight into catalytic mechanism of DapE enzymes as well as a structural foundation that is critical for the rational design of DapE inhibitors.     

Transfer of nitric oxide from nitrovasodilators to free thiols--evidence of two distinct stages

Two distinct stages, which can be monitored spectrophotometrically, have been observed for the first time in multiple reactions between thiol, such as l-cysteine and some well-known nitrovasodilators, namely S-nitroso-N-acetyl-d,l-penicillamine (SNAP), S-nitrosoglutathione (GSNO), and S-nitrosocaptopril (SNOCap) in aqueous solution (in the presence of EDTA). The first part of the reaction occurs at stopped-flow time scale ( approximately 10(-2)s(-1)) in one single step and has been found to be transnitrosation; followed by a slow decomposition of the products of the transnitrosation reaction with the formation of a variety of nitrogen products. Reactivity with regard to the first stage occurs in the order GSNO>SNAP>SNOCap. The second stage shows first-order rate constants of the order 10(-4)s(-1), although some degree of complexity exists in elucidating an accurate rate equation with which a comparative study could be done.     

Synergism between hydrogen sulfide (H(2)S) and nitric oxide (NO) in vasorelaxation induced by stonustoxin (SNTX), a lethal and hypotensive protein factor isolated from stonefish Synanceja horrida venom

Stonustoxin (SNTX) is a 148 kDa, dimeric, hypotensive and lethal protein factor isolated from the venom of the stonefish Synanceja horrida. SNTX (10-320 ng/ml) progressively causes relaxation of endothelium-intact, phenylephrine (PE)-precontracted rat thoracic aortic rings. The SNTX-induced vasorelaxation was inhibited by L-N(G)-nitro arginine methyl ester (L-NAME), suggesting that nitric oxide (NO) contributes to the SNTX-induced response. Interestingly, D, L-proparglyglycine (PAG) and beta-cyano-L-alanine (BCA), irreversible and competitive inhibitors of cystathionine-gamma-lyase (CSE) respectively, also inhibited SNTX-induced vasorelaxation, indicating that H(2)S may also play a part in the effect of SNTX. The combined use of L-NAME with PAG or BCA showed that H(2)S and NO act synergistically in effecting SNTX-induced vasorelaxation.     

Effect of 24-epibrassinolide on the photosynthetic activity of radish plants under cadmium stress

The present study was conducted to study the effect of 24-epibrassinolide (EBL) on changes of plant growth, net photosynthetic rate, carbonic anhydrase (E.C. 4.2.1.1) and nitrate reductase (E.C.1.6.6.1) activities in the leaves of Raphanus sativus L. under the influence of cadmium (Cd) stress. Cd reduced plant growth, photosynthetic pigment levels, net photosynthetic rate and the activities of carbonic anhydrase and nitrate reductase. However seed application of EBL reduced the toxic effect of Cd on plant growth, pigment content, photosynthesis and enzyme activities. The studies clearly demonstrated the ameliorating effect of 24-epibrassinolide in mitigating the toxicity of Cd in plants.     

Reduction of Nomega-hydroxy-L-arginine to L-arginine by pig liver microsomes, mitochondria, and human liver microsomes

Nomega-Hydroxy-L-arginine, the intermediate in nitric oxide formation from L-arginine catalyzed by NO synthase, can be released into the extracellular space. It has been suggested that it can circulate and exert paracrine effects. Since it cannot only be used as substrate by NO synthases, but can also be oxidized by cytochrome P450 and other hemoproteins in a superoxide-dependent manner, it has been proposed that it can serve as NO donor. In the present study, the in vitro reduction of Nomega-hydroxy-L-arginine was examined. Pig and human liver microsomes as well as pig liver mitochondria were capable of reducing Nomega-hydroxy-L-arginine to L-arginine in an oxygen-insensitive enzymatic reaction. These results demonstrate that this metabolic pathway has to be considered when suggesting Nomega-hydroxy-L-arginine as NO-precursor. The reconstituted liver microsomal system of a pig liver CYP2D enzyme, the benzamidoxime reductase, was unable to replace microsomes to produce L-arginine from Nomega-hydroxy-L-arginine.     

The Pathway of Product Release from the R State of Aspartate Transcarbamoylase

The pathway of product release from the R state of aspartate transcarbamoylase (ATCase; EC 2.1.3.2, aspartate carbamoyltransferase) has been determined here by solving the crystal structure of Escherichia coli ATCase locked in the R quaternary structure by specific introduction of disulfide bonds. ATCase displays ordered substrate binding and product release, remaining in the R state until substrates are exhausted. The structure reported here represents ATCase in the R state bound to the final product molecule, phosphate. This structure has been difficult to obtain previously because the enzyme relaxes back to the T state after the substrates are exhausted. Hence, cocrystallizing the wild-type enzyme with phosphate results in a T-state structure. In this structure of the enzyme trapped in the R state with specific disulfide bonds, we observe two phosphate molecules per active site. The position of the first phosphate corresponds to the position of the phosphate of carbamoyl phosphate (CP) and the position of the phosphonate of N-phosphonacetyl-l-aspartate. However, the second, more weakly bound phosphate is bound in a positively charged pocket that is more accessible to the surface than the other phosphate. The second phosphate appears to be on the path that phosphate would have to take to exit the active site. Our results suggest that phosphate dissociation and CP binding can occur simultaneously and that the dissociation of phosphate may actually promote the binding of CP for more efficient catalysis.     

The Hypocrea jecorina (syn. Trichoderma reesei) lxr1 gene encodes a d-mannitol dehydrogenase and is not involved in l-arabinose catabolism

The Hypocrea jecorina LXR1 was described as the first fungal l-xylulose reductase responsible for NADPH dependent reduction of l-xylulose to xylitol in l-arabinose catabolism. Phylogenetic analysis now reveals that LXR1 forms a clade with fungal d-mannitol 2-dehydrogenases. Lxr1 and the orthologous Aspergillus nigermtdA are not induced by l-arabinose but expressed at low levels during growth on different carbon sources. Deletion of lxr1 does not affect growth on l-arabinose and l-xylulose reductase activity remains unaltered whereas d-mannitol 2-dehydrogenase activities are reduced. We conclude that LXR1 is a d-mannitol 2-dehydrogenase and that a true LXR1 is still awaiting discovery.     

Inducible nitric oxide synthase aggresome formation is mediated by nitric oxide

Nitric oxide (NO) generated by inducible NO synthase (iNOS) contributes critically to inflammatory injury and host defense. While previously thought as a soluble protein, iNOS was recently reported to form aggresomes inside cells. But what causes iNOS aggresome formation is unknown. Here we provide evidence demonstrating that iNOS aggresome formation is mediated by its own product NO. Exposure to inflammatory stimuli (lipopolysaccharide and interferon-γ) induced robust iNOS expression in mouse macrophages. While initially existing as a soluble protein, iNOS progressively formed protein aggregates as a function of time. Aggregated iNOS was inactive. Treating the cells with the NOS inhibitor N-nitro-l-arginine methyl ester (L-NAME) blocked NO production from iNOS without affecting iNOS expression. However, iNOS aggregation in cells was prevented by L-NAME. The preventing effect of NO blockade on iNOS aggresome formation was directly observed in GFP-iNOS-transfected cells by fluorescence imaging. Moreover, iNOS aggresome formation could be recaptured by adding exogenous NO to L-NAME-treated cells. These studies demonstrate that iNOS aggresome formation is caused by NO. The finding that NO induces iNOS aggregation and inactivation suggests aggresome formation as a feedback inhibition mechanism in iNOS regulation.     

Mitochondrial metabolic states and membrane potential modulate mtNOS activity

The mitochondrial metabolic state regulates the rate of NO release from coupled mitochondria: NO release by heart, liver and kidney mitochondria was about 40-45% lower in state 3 (1.2, 0.7 and 0.4 nmol/min mg protein) than in state 4 (2.2, 1.3 and 0.7 nmol/min mg protein). The activity of mtNOS, responsible for NO release, appears driven by the membrane potential component and not by intramitochondrial pH of the proton motive force. The intramitochondrial concentrations of the NOS substrates, l-arginine (about 310 μM) and NADPH (1.04-1.78 mM) are 60-1000 times higher than their K_M values. Moreover, the changes in their concentrations in the state 4-state 3 transition are not enough to explain the changes in NO release. Nitric oxide release was exponentially dependent on membrane potential as reported for mitochondrial H₂O₂ production [S.S. Korshunov, V.P. Skulachev, A.A. Satarkov, High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria. FEBS Lett. 416 (1997) 15-18.]. Agents that decrease or abolish membrane potential minimize NO release while the addition of oligomycin that produces mitochondrial hyperpolarization generates the maximal NO release. The regulation of mtNOS activity, an apparently voltage-dependent enzyme, by membrane potential is marked at the physiological range of membrane potentials.     

Site-directed mutagenesis studies of acetylglutamate synthase delineate the site for the arginine inhibitor

N-acetyl-l-glutamate synthase (NAGS), the first enzyme of bacterial/plant arginine biosynthesis and an essential activator of the urea cycle in animals, is, respectively, arginine-inhibited and activated. Site-directed mutagenesis of recombinant Pseudomonas aeruginosa NAGS (PaNAGS) delineates the arginine site in the PaNAGS acetylglutamate kinase-like domain, and, by extension, in human NAGS. Key residues for glutamate binding are identified in the acetyltransferase domain. However, the acetylglutamate kinase-like domain may modulate glutamate binding, since one mutation affecting this domain increases the K_m for glutamate. The effects on PaNAGS of two mutations found in human NAGS deficiency support the similarity of bacterial and human NAGSs despite their low sequence identity.     

First record of L-quebrachitol in Allophylus edulis (Sapindaceae)

Allophylus edulis, commonly called ‘Chal chal’, is a member of the Sapindaceae occurring in the Uruguayan and Brazilian native flora. During the phytochemical analysis of two Chal chal specimens from two well-differentiated geographical zones (Assis, São Paulo, Brazil, and Santa Lucía, Canelones, Uruguay), considerable amounts of l-quebrachitol were isolated from both samples. The isolation was carried out from the ethanolic twig extracts obtained by maceration of both vegetal samples. White easily distinguishable crystals were mechanically separated, washed, and characterized by 1D and 2D NMR experiments and by MS data. Such techniques confirmed that the crystals isolated from sources collected in both countries resulted in the same compound, l-quebrachitol, a natural product not previously reported for this species and one that has been investigated as a sugar substitute for diabetics. Worthy of note, the content of l-quebrachitol in A. edulis may be the chemical basis to explain its ethnobotanical uses, since infusions of this plant are used to treat diabetes in the practice of local traditional medicine.     

Thermodynamic determination of the binding constants of angiotensin-converting enzyme inhibitors by a displacement method

Somatic angiotensin I-converting enzyme (s-ACE) plays a central role in blood pressure regulation and has been the target of most antihypertensive drugs. A displacement isothermal titration calorimetry method has been used to accurately determine the binding constant of three strong s-ACE inhibitors. Under the experimental conditions studied in this work, the relative potency of the inhibitors was determined to be enalaprilat>lisinopril>captopril. We analyze the thermodynamic behaviour of the binding process using the new structural information provided by the ACE structures, as well as the conformational changes that occur upon binding.     

A new route to dTDP-6-deoxy-l-talose and dTDP-L-rhamnose: dTDP-L-rhamnose 4-epimerase in Burkholderia thailandensis

dTDP-l-rhamnose (dTDP-Rha)-synthesizing dTDP-6-deoxy-l-lyxo-4-hexulose reductase (4-KR) and dTDP-Rha 4-epimerase were characterized from Burkholderia thailandensis E264 by utilizing rmlD_Bth (BTH_I1472) and wbiB_Bth (BTH_I1476), respectively. Incubation of the recombinant WbiB_Bth with RmlA/RmlB/RmlC/Tal, which has previously been shown to generate dTDP-6-deoxy-l-talose (dTDP-6dTal) from α-d-glucose-1-phosphate, dTTP, and NADPH, produced dTDP-Rha. ¹H NMR measurements confirmed that both RmlA/RmlB/RmlC/Tal/WbiB_Bth and RmlA/RmlB/RmlC/RmlD produced dTDP-Rha. WbiB_Bth alone produced dTDP-Rha when incubated with dTDP-6dTal. This is the first report to demonstrate epimerase activity interconverting between dTDP-Rha and dTDP-6dTal.     

Ascorbic acid synthesis due to L-gulono-1,4-lactone oxidase expression enhances NO production in endothelial cells

As a primary antioxidant, ascorbic acid (AA) provides beneficial effects for vascular health mitigating oxidative stress and endothelial dysfunction. However, the association of intracellular AA with NO production occurring inside the endothelial cells remains unclear. In the present study, we addressed this issue by increasing intracellular AA directly through de novo synthesis. To restore AA synthesis pathway, bovine aortic endothelial cells were transfected with the plasmid vector encoding L-gulono-1,4-lactone oxidase (GULO, EC 1.1.3.8), the missing enzyme converting L-gulono-1,4-lactone (GUL) to AA. Functional expression of GULO was verified by Western blotting and in vitro enzyme activity assay. GULO expression alone did not lead to AA synthesis but the supply of GUL resulted in a marked increase of intracellular AA. When the cells were stimulated with calcium ionophore, A23187, NO production was more active in the GULO-expressing cells supplied with GUL, in comparison with the cells without GULO expression or without GUL supply, indicating that intracellular AA regulated NO production. Enhancement of NO production by intracellular AA was further verified in aortic endothelial cells obtained from eNOS knockout mice that were cotransfected with eNOS and GULO constructs. GULO-dependent AA synthesis also elevated intracellular tetrahydrobiopterin content, implicating that this essential cofactor of endothelial nitric oxide synthase (eNOS) might mediate the AA effect. The present study strongly suggests that intracellular AA plays critical roles in vascular physiology through enhancing endothelial NO production.     

Substrate specificity of human glutamine transaminase K as an aminotransferase and as a cysteine S-conjugate beta-lyase

Rat kidney glutamine transaminase K (GTK) exhibits broad specificity both as an aminotransferase and as a cysteine S-conjugate β-lyase. The β-lyase reaction products are pyruvate, ammonium and a sulfhydryl-containing fragment. We show here that recombinant human GTK (rhGTK) also exhibits broad specificity both as an aminotransferase and as a cysteine S-conjugate β-lyase. S-(1,1,2,2-Tetrafluoroethyl)-l-cysteine is an excellent aminotransferase and β-lyase substrate of rhGTK. Moderate aminotransferase and β-lyase activities occur with the chemopreventive agent Se-methyl-l-selenocysteine. l-3-(2-Naphthyl)alanine, l-3-(1-naphthyl)alanine, 5-S-l-cysteinyldopamine and 5-S-l-cysteinyl-l-DOPA are measurable aminotransferase substrates, indicating that the active site can accommodate large aromatic amino acids. The α-keto acids generated by transamination/l-amino acid oxidase activity of the two catechol cysteine S-conjugates are unstable. A slow rhGTK-catalyzed β-elimination reaction, as measured by pyruvate formation, was demonstrated with 5-S-l-cysteinyldopamine, but not with 5-S-l-cysteinyl-l-DOPA. The importance of transamination, oxidation and β-elimination reactions involving 5-S-l-cysteinyldopamine, 5-S-l-cysteinyl-l-DOPA and Se-methyl-l-selenocysteine in human tissues and their biological relevance are discussed.     

D-Erythroascorbic acid activates cyanide-resistant respiration in Candida albicans

Higher plants, protists and fungi possess cyanide-resistant respiratory pathway, which is mediated by alternative oxidase (AOX). The activity of AOX has been found to be dependent on several regulatory mechanisms including gene expression and posttranslational regulation. In the present study, we report that the presence of cyanide in culture medium remarkably retarded the growth of alo1/alo1 mutant of Candida albicans, which lacks d-arabinono-1,4-lactone oxidase (ALO) that catalyzes the final step of d-erythroascorbic acid (EASC) biosynthesis. Measurement of respiratory activity and Western blot analysis revealed that increase in the intracellular EASC level induces the expression of AOX in C. albicans. AOX could still be induced by antimycin A, a respiratory inhibitor, in the absence of EASC, suggesting that several factors may act in parallel pathways to induce the expression of AOX. Taken together, our results suggest that EASC plays important roles in activation of cyanide-resistant respiration in C. albicans.     

Leptin improves membrane fluidity of erythrocytes in humans via a nitric oxide-dependent mechanism--an electron paramagnetic resonance investigation

Abnormalities in physical properties of the cell membranes may underlie the defects that are strongly linked to hypertension, stroke, and other cardiovascular diseases. Recently, there has been an indication that leptin, the product of the human obesity gene, actively participates not only in the metabolic regulations but also in the control of cardiovascular functions. In the present study, to assess the role of leptin in the regulation of membrane properties, the effects of leptin on membrane fluidity of erythrocytes in humans are examined. The membrane fluidity of erythrocytes in healthy volunteers by means of an electron paramagnetic resonance (EPR) and spin-labeling method is determined. In an in vitro study, leptin decreased the order parameter (S) for 5-nitroxide stearate (5-NS) and the peak height ratio (ho/h-1) for 16-NS obtained from EPR spectra of erythrocyte membranes in a dose-dependent manner in healthy volunteers. The finding indicated that leptin increased the membrane fluidity and improved the microviscosity of erythrocytes. The effect of leptin on the membrane fluidity was significantly potentiated by the nitric oxide (NO) donors, L-arginine and S-nitroso-N-acetylpenicillamine (SNAP), and a cyclic guanosine monophosphate (cGMP) analog, 8-bromo-cGMP. In contrast, the change evoked by leptin was significantly attenuated in the presence of the NO synthase inhibitors, N(G)-nitro-L-arginine-methyl-ester (L-NAME) and asymmetric dimethyl-L-arginine (ADMA). The results of the present study showed that leptin increased the membrane fluidity and improved the rigidity of cell membranes to some extent via an NO- and cGMP-dependent mechanism. Furthermore, the data also suggest that leptin might have a crucial role in the regulation of rheological behavior of erythrocytes and microcirculation in humans.     

Glucagon-like peptide-1 relaxes gastric antrum through nitric oxide in mice

Glucagon-like-peptide-1 (GLP-1) is a proglucagon-derived peptide expressed in the intestinal enteroendocrine-L cells and released after meal ingestion. GLP-1 reduces postprandial glycemia not only by its hormonal effects, but also by its inhibitory effects on gastrointestinal motility. Recently, we showed that GLP-1 acts in the enteric nervous system of mouse intestine. Therefore our working hypothesis was that GLP-1 may have also a direct influence on the gastric mechanical activity since the major part of experimental studies about its involvement in the regulation of gastric motility have been conducted in in vivo conditions. The purposes of this study were (i) to examine exogenous GLP-1 effects on mouse gastric mechanical activity using isolated whole stomach; (ii) to clarify the regional activity of GLP-1 using circular muscular strips from gastric fundus or antrum; (iii) to analyze the mechanism of action underlying the observed effects; (iv) to verify regional differences of GLP-1 receptors (GLP-1R) expression by RT-PCR. In the whole stomach GLP-1 caused concentration-dependent relaxation significantly anatagonized by exendin (9-39), an antagonist of GLP-1R and abolished by tetrodotoxin (TTX) or N_ω-nitro-l-arginine methyl ester (l-NAME), inhibitor of nitric oxide (NO) synthase. GLP-1 was without any effect in fundic strips, but it induced concentration-dependent relaxation in carbachol-precontracted antral strips. The effect was abolished by TTX or l-NAME. RT-PCR analysis revealed a higher expression of GLP-1R mRNA in antrum than in fundus. These results suggest that exogenous GLP-1 is able to reduce mouse gastric motility by acting peripherally in the antral region, through neural NO release.     

Diazepam effects on carrageenan-induced inflammatory paw edema in rats: role of nitric oxide

High doses of diazepam (10.0-20.0 mg/kg) were shown to reduce the volume of acute inflammatory paw edema in rats as a response to carrageenan administration. This effect was attributed to an action of diazepam on the peripheral-type benzodiazepine receptor (PBR) present in the adrenal and/or immune/inflammatory cells. The present study was undertaken to analyze the involvement of nitric oxide (NO) on the effects of diazepam on carrageenan-induced paw edema in rats (CIPE) and to look for the presence of PBR and inducible/constitutive NO synthases (NOS) on slices taken from the inflamed paws of diazepam-treated rats. For that, an acute inhibition of NO biosynthesis was achieved using 50.0 mg/kg No mega-nitro-L-arginine (L-NAME), L-arginine (300.0 mg/kg), the true precursor of NO, and D-arginine (300.0 mg/kg), its false substrate, were also used. The following results were obtained: (1) diazepam (10.0 and 20.0 mg/kg) decreased CIPE values in a dose- and time-dependent way; (2) diazepam effects on CIPE were increased by L-NAME pretreatment; (3) treatment with L-arginine but not with D-arginine reverted at least in part the decrements of CIPE values observed after diazepam administration; (4) PBR were found in endothelial and inflammatory cells that migrated to the inflammatory site at the rat paw; (5) confocal microscopy showed the presence of both PBR and NOS in endothelial and inflammatory cells taken from inflamed paw tissues of rats treated with diazepam a finding not observed in tissues provided from rats treated with diazepam's control solution. These results suggest an important role for NO on the effects of diazepam on CIPE. Most probably, these effects reflect a direct action of diazepam on PBR present in the endothelium of the microvascular ambient and/or on immune/inflammatory cells. An action like that would lead, among other factors, to a decrease in NO, generated by NO synthase, and thus in the mechanisms responsible for CIPE.