ABSTRACT
The present study was undertaken to determine the mechanism of antibacterial activity of a polyphenolic fraction, composed of mainly catechin and isorhamnetin, previously isolated from Kombucha, a 14-day fermented beverage of sugared black tea, against the enteropathogen Vibrio cholerae N16961. Bacterial growth was found to be seriously impaired by the polyphenolic fraction in a dose-dependent manner. Scanning Electron Microscopy demonstrated morphological alterations in bacterial cells when exposed to the polyphenolic fraction in a concentration-dependent manner. Permeabilization assays confirmed that the fraction disrupted bacterial membrane integrity in both time- and dose-dependent manners, which were proportional to the production of intracellular reactive oxygen species (ROS). Furthermore, each of the polyphenols catechin and isorhamnetin showed the ability to permeate bacterial cell membranes by generating oxidative stress, thereby suggesting their role in the antibacterial potential of Kombucha. Thus, the basic mechanism of antibacterial activity of the Kombucha polyphenolic fraction against V. cholerae involved bacterial membrane permeabilization and morphological changes, which might be due to the generation of intracellular ROS. To the best of our knowledge, this is the first report on the investigation of antibacterial mechanism of Kombucha, which is mostly attributed to its polyphenolic content. SIGNIFICANCE AND IMPACT OF THE STUDY: The emergence of multidrug-resistant Vibrio cholerae strains has hindered an efficient anti-Vibrio therapy. This study has demonstrated the membrane damage-mediated antibacterial mechanism of Kombucha, a popular fermented beverage of sugared tea, which is mostly attributed to its polyphenolic content. This study also implies the exploitation of Kombucha as a potential new source of bioactive polyphenols against V. cholerae.
Subject(s)
Anti-Bacterial Agents/pharmacology , Catechin/pharmacology , Kombucha Tea/analysis , Polyphenols/pharmacology , Quercetin/analogs & derivatives , Vibrio cholerae/drug effects , Camellia sinensis/chemistry , Cell Membrane/metabolism , Cell Membrane Permeability/drug effects , Fermentation , Oxidative Stress , Quercetin/pharmacology , Reactive Oxygen Species/metabolism , Tea , Vibrio cholerae/growth & developmentABSTRACT
An avirulent, live transconjugant Shigella hybrid (LTSHΔstx) strain was constructed in our earlier study by introducing a plasmid vector, pPR1347, into a Shiga toxin gene deleted Shigella dysenteriae 1. Three successive oral administrations of LTSHΔstx to female adult mice produced comprehensive passive heterologous protection in their offspring against challenge with wild-type shigellae. Production of NO and different cytokines such asIL-12p70, IL-1ß and IL-23 in peritoneal mice macrophages indicated that LTSHΔstx induced innate and adaptive immunity in mice. Furthermore, production of IFN-γ, IL-10 and IL-17 in LTSH-primed splenic CD4+ T cell suggested that LTSHΔstx may induce Th1 and Th17 cell-mediated immune responses. Exponential increase of the serum IgG and IgA titre against whole shigellae was observed in immunized adult mice during and after the immunization with the highest peak on day 35. Antigen-specific sIgA was also determined from intestinal lavage of immunized mice. The stomach extracts of neonates from immunized mice, mainly containing mother's milk, contained significant levels of anti-LTSHΔstx immunoglobulin. These studies suggest that the LTSHΔstx could be a new live oral vaccine candidate against shigellosis in the near future.
Subject(s)
Shigella/immunology , Th1 Cells/immunology , Th17 Cells/immunology , Administration, Oral , Animals , Animals, Newborn , Antibodies, Bacterial/biosynthesis , Antigens, Bacterial/genetics , Bacterial Vaccines/administration & dosage , Bacterial Vaccines/genetics , Bacterial Vaccines/immunology , Conjugation, Genetic , Disease Models, Animal , Dysentery, Bacillary/immunology , Dysentery, Bacillary/microbiology , Dysentery, Bacillary/prevention & control , Female , Gene Deletion , Genes, Bacterial , Immunity, Cellular , Immunization, Passive , Male , Mice , Mice, Inbred BALB C , Shiga Toxin/genetics , Shigella/genetics , Shigella/pathogenicity , Shigella dysenteriae/genetics , Shigella dysenteriae/immunology , Shigella dysenteriae/pathogenicity , Species Specificity , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/genetics , Vaccines, Attenuated/immunology , Virulence/geneticsABSTRACT
The Acetobacteraceae family of the class Alpha Proteobacteria is comprised of high sugar and acid tolerant bacteria. The Acetic Acid Bacteria are the economically most significant group of this family because of its association with food products like vinegar, wine etc. Acetobacteraceae are often hard to culture in laboratory conditions and they also maintain very low abundances in their natural habitats. Thus identification of the organisms in such environments is greatly dependent on modern tools of molecular biology which require a thorough knowledge of specific conserved gene sequences that may act as primers and or probes. Moreover unconserved domains in genes also become markers for differentiating closely related genera. In bacteria, the 16S rRNA gene is an ideal candidate for such conserved and variable domains. In order to study the conserved and variable domains of the 16S rRNA gene of Acetic Acid Bacteria and the Acetobacteraceae family, sequences from publicly available databases were aligned and compared. Near complete sequences of the gene were also obtained from Kombucha tea biofilm, a known Acetobacteraceae family habitat, in order to corroborate the domains obtained from the alignment studies. The study indicated that the degree of conservation in the gene is significantly higher among the Acetic Acid Bacteria than the whole Acetobacteraceae family. Moreover it was also observed that the previously described hypervariable regions V1, V3, V5, V6 and V7 were more or less conserved in the family and the spans of the variable regions are quite distinct as well.
Subject(s)
Acetobacteraceae/genetics , Genes, Bacterial , RNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , Acetic Acid/metabolism , Acetobacteraceae/classification , Acetobacteraceae/metabolism , Base Sequence , Chromosome Mapping , Conserved Sequence , Genetic Variation , Molecular Sequence Data , Phylogeny , Sequence AlignmentABSTRACT
AIMS: Mercury compounds are highly toxic to all types of living cells. Isolated yeast strains of Rhodotorula rubra showed high and low resistance pattern towards mercury and organomercurial compounds. To investigate the basis of differential sensitivity of these two types of strains, glucose utilization was measured in the presence of mercury compounds. METHODS AND RESULTS: Glucose utilization process remained unaffected in resting cells of highly Hg(2+)-resistant strain in the presence of HgCl(2) but not in the presence of phenylmercuric acetate and thimerosal. However, HgCl(2) significantly affected glucose utilization in the case of low-resistant cells. The Hg-retaining ability of the cell wall of highly Hg(2+)-resistant yeast strain was greater than that of the weakly Hg(2+)-resistant strain. The spheroplast-bound Hg(2+) was also significantly less in the highly Hg(2+)-resistant strain than in the weakly Hg(2+)-resistant strain. CONCLUSIONS: Glucose uptake machinery was not affected in the presence of toxic metal ions in the case of high-resistant strains. But in the case of low Hg(2+)-resistant strain, glucose transport system may be affected either by inactivation of sensor proteins containing -SH group associated with glucose uptake. SIGNIFICANCE AND IMPACT OF THE STUDY: Cell wall of mercury-resistant yeast cells may play an important role in heavy metal bioremediation process.
Subject(s)
Drug Resistance, Microbial , Glucose/metabolism , Mercury/pharmacology , Organomercury Compounds/pharmacology , Saccharomyces cerevisiae/drug effects , Cell Wall/drug effects , Mercuric Chloride/pharmacology , Mycology/methods , Phenylmercuric Acetate/pharmacology , Saccharomyces cerevisiae/metabolism , Spheroplasts/drug effects , Thimerosal/pharmacology , Time FactorsABSTRACT
Environmental and occupational lead pollution is a common problem in both developing and industrialised countries. Both hepatotoxicity and nephrotoxicity are known to occur in persons with exposure to heavy metals. We studied both liver function and renal function and blood lead concentraton in random population sample of 372 men (age range, 24 to 55 years). In all the subjects we measured both liver and renal function tests and both blood lead and urinary concentration of lead. Raised blood and urinary lead concentrations were associated with moderate changes in liver function and abnormal renal function, reflected in decrease of albumin and increased levels of liver enzymes and raised urea and creatinine concentrations, and with a reduction in creatinine clearance rate as compared to apparently normal subjects. These findings emphasis the importance of measurement of blood lead concentrations in adults in the genereal population to combat the effects of lead toxicity before the clinical signs predominate.
Subject(s)
Chemical and Drug Induced Liver Injury , Kidney Diseases/chemically induced , Lead/blood , Lead/toxicity , Occupational Exposure , Adult , Case-Control Studies , Creatinine/blood , Cross-Sectional Studies , Humans , Kidney/drug effects , Kidney/pathology , Liver/drug effects , Liver/pathology , Male , Middle Aged , Serum Albumin , Urea/bloodABSTRACT
Nitric oxide synthases (NOSs) catalyze the formation of nitric oxide from L-arginine. We purified the heme containing, tetrahydrobiopterin-free, oxygenase domain of rat neuronal nitric oxide synthase (nNOSox) overexpressed in Escherichia coli. We found catalase activity in nNOSox. This is significant because H(2)O(2) may also be a product of nitric oxide synthases. We found H(2)O(2) assisted product formation from N-hydroxy-L-arginine and even from L-arginine both in the presence and in absence of tetrahydrobiopterin. We propose how heme moiety of the oxygenase domain alone is sufficient to carry out both steps of the NOS catalysis.
Subject(s)
Catalase/chemistry , Nerve Tissue Proteins/chemistry , Nitric Oxide Synthase/chemistry , Oxygenases/chemistry , Animals , Arginine , Catalase/metabolism , Escherichia coli , Hydrogen Peroxide , Nerve Tissue Proteins/metabolism , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type I , Oxygenases/metabolism , RatsABSTRACT
The oxygenase domain of the inducible nitric oxide synthase (iNOSox; residues 1-498) is a dimer that binds heme, L-arginine and tetrahydrobiopterin (H(4)B) and is the site for nitric oxide synthesis. We examined an N-terminal segment that contains a beta-hairpin hook, a zinc ligation center and part of the H(4)B-binding site for its role in dimerization, catalysis, and H(4)B and substrate interactions. Deletion mutagenesis identified the minimum catalytic core and indicated that an intact N-terminal beta-hairpin hook is essential. Alanine screening mutagenesis of conserved residues in the hook revealed five positions (K82, N83, D92, T93 and H95) where native properties were perturbed. Mutants fell into two classes: (i) incorrigible mutants that disrupt side-chain hydrogen bonds and packing interactions with the iNOSox C-terminus (N83, D92 and H95) and cause permanent defects in homodimer formation, H(4)B binding and activity; and (ii) reformable mutants that destabilize interactions of the residue main chain (K82 and T93) with the C-terminus and cause similar defects that were reversible with high concentrations of H(4)B. Heterodimers comprised of a hook-defective iNOSox mutant subunit and a full-length iNOS subunit were active in almost all cases. This suggests a mechanism whereby N-terminal hooks exchange between subunits in solution to stabilize the dimer.
Subject(s)
Biopterins/analogs & derivatives , Nitric Oxide Synthase/chemistry , Nitric Oxide Synthase/metabolism , Pterins/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Binding Sites , Biopterins/metabolism , Cattle , Dimerization , Drosophila , Humans , Kinetics , Mice , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Nitric Oxide Synthase Type II , Point Mutation , Protein Structure, Secondary , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Deletion , Sequence Homology, Amino Acid , SpectrophotometryABSTRACT
Nitric oxide synthase (NOS) is a heme protein that catalyzes the oxygenation of L-arginine in the presence of NADPH to form nitric oxide, L-citrulline and NADP+, and proceeds via two partial reactions: 1) L-Arginine --> NG-hydroxy-L-arginine 2) NG-Hydroxy-L-arginine --> L-citrulline + nitric oxide Calmodulin, FAD, FMN and tetrahydrobiopterin are required for both reactions. Reactions 1 and 2 require the input of 2 and 1 electron equivalents, respectively. Under normal multiple turnover conditions, these electrons are ultimately derived from NADPH. We previously reported that NOS contains an endogenous reductant that, in the absence of NADPH, can support the single-turnover oxygenation of L-arginine to NG-hydroxy-L-arginine and a relatively small amount of L-citrulline [Campos, K. L., Giovanelli, J., and Kaufman, S. (1995) J. Biol. Chem. 270, 1721-1728]. This reductant has now been identified as the stable flavin semiquinone free radical (FSQ). Its oxidation appears to be coupled to the formation of NG-hydroxy-L-arginine and L-citrulline. The rate of FSQ oxidation is two orders of magnitude slower than the flux of electrons from NADPH through NOS during normal turnover of the enzyme, indicating that FSQ is not the proximal electron donor for heme under these conditions.
Subject(s)
Arginine/analogs & derivatives , Arginine/metabolism , Flavin-Adenine Dinucleotide/analogs & derivatives , Nitric Oxide Synthase/metabolism , Nitric Oxide/biosynthesis , Oxygen/metabolism , Arginine/biosynthesis , Citrulline/metabolism , Electron Spin Resonance Spectroscopy , Flavin-Adenine Dinucleotide/metabolism , Kinetics , Oxidation-ReductionABSTRACT
Endothelial nitric-oxide synthase (eNOS) is targeted to caveoli through interaction with caveolin-1 (cav-1). cav-1 binding to a consensus site in the eNOS oxygenase domain is proposed to antagonize calmodulin (CaM) binding and thereby inhibit eNOS nitric oxide (NO) synthesis. To study the mechanism, we examined how cav-1 scaffolding domain peptide (amino acids 82-101; cav-1P) would affect NO synthesis, NADPH oxidation, cytochrome c reduction, and ferricyanide reduction by full-length eNOS or its isolated oxygenase and reductase domains. Cav-1P equivalently inhibited NO synthesis and NADPH oxidation by full-length eNOS in a manner reversible by CaM but did not affect NADPH-independent NO synthesis by full-length eNOS or its oxygenase domain, indicating inhibition required the reductase domain. Similar concentrations of cav-1P inhibited cytochrome c reduction by full-length eNOS or the reductase domain (amino acids 492-1205) in a CaM-reversible manner, indicating cav-1P interaction with reductase or full-length eNOS are equivalent. Ferricyanide reduction was unaffected by cav-1P in all cases. Immunoblotting showed that full-length eNOS, eNOS oxygenase, and eNOS reductase all bound to an immobilized glutathione S-transferase-cav-1 fusion protein. Thus, cav-1 interacts independently with both oxygenase and reductase domains of eNOS. The reductase interaction occurs independent of a cav-1 binding motif, is CaM-reversible, and is of sufficient affinity to match cav-1P inhibition of NO synthesis by full-length eNOS. We propose that cav-1 binding to eNOS reductase compromises its ability to bind CaM and to donate electrons to the eNOS heme, thereby inhibiting NO synthesis.
Subject(s)
Caveolins , Membrane Proteins/metabolism , Nitric Oxide Synthase/metabolism , Oxidoreductases/metabolism , Amino Acid Sequence , Catalysis , Caveolin 1 , Heme/metabolism , Molecular Sequence Data , Nitric Oxide/biosynthesis , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/chemistry , Nitric Oxide Synthase Type IIIABSTRACT
Nitric oxide synthases (NOS) are heme proteins, closely related to cytochromes P450, that catalyze oxidation of l-arginine (l-Arg) to nitric oxide (NO) and citrulline. To get further insight into their active site, we have studied the ability of recombinant mouse inducible NOS (iNOS) and rat brain neuronal NOS (nNOS), and of their oxygenase domains (iNOSoxy and nNOSoxy), to form Fe(II)-nitrosoalkane complexes. In the absence of BH4, iNOSoxy, nNOSoxy, and full-length iNOS readily form complexes characterized by Soret peaks around 448 nm, after reaction with various nitroalkanes and sodium dithionite. These complexes displayed physicochemical characteristics very similar to those of previously reported microsomal cytochrome P450-Fe(II)-nitrosoalkane complexes: (i) a Soret peak around 450 nm, (ii) a clear stability in the presence of CO, and (iii) a fast destruction upon oxidation of the iron by ferricyanide. Thus, in the absence of l-Arg and BH4, NOSs Fe(II) appear to be largely opened to even large R-NO ligands with R = cyclohexyl or p-Cl-C6H4-CH2CH(CH3) for instance, in a manner similar to microsomal P450s Fe(II). As expected, the presence of l-Arg inhibits the formation of NOSs Fe(II)-RNO complexes. More surprisingly, the presence of BH4 also strongly inhibits the formation of the NOSs Fe(II) complexes even with the smallest nitrosoalkane ligand, CH3NO (IC50 values of 0.5 and 4 microM for nNOSoxy and iNOSoxy, respectively). Accordingly, recombinant full-length nNOS containing BH4 and l-Arg is completely unable to form Fe(II)-nitrosoalkane complexes, even with CH3NO. These results suggest that, in the absence of l-Arg and BH4, the distal pocket of NOSs Fe(II) is largely opened even to bulky ligands, in a manner similar to that of microsomal cytochromes P450. On the contrary, the distal heme pocket of iNOS and nNOS seems to be closed after binding of l-Arg and BH4, particularly in the Fe(II) state. This results in a highly restricted access for Fe(II) ligands, except very small ones such as CO, NO, and O2. Such effects of BH4 in controlling the size of the distal heme pocket of NOS Fe(II) correspond to a new role of biopterins in biological systems.
Subject(s)
Alkanes/metabolism , Biopterins/analogs & derivatives , Ferrous Compounds/metabolism , Nitric Oxide Synthase/metabolism , Nitroso Compounds/metabolism , Animals , Biopterins/metabolism , Dimerization , Macromolecular Substances , Methane/analogs & derivatives , Methane/metabolism , Mice , Nitric Oxide Synthase Type I , Nitric Oxide Synthase Type II , Nitroparaffins/metabolism , Protein Conformation , Rats , Spectrophotometry, UltravioletABSTRACT
Calmodulin (CaM) binding activates neuronal nitric-oxide synthase (nNOS) catalytic functions and also up-regulates electron transfer into its flavin and heme centers. Here, we utilized seven tight binding CaM-troponin C chimeras, which variably activate nNOS NO synthesis to examine the relationship between CaM domain structure, activation of catalytic functions, and control of internal electron transfer at two points within nNOS. Chimeras that were singly substituted with troponin C domains 4, 3, 2, or 1 were increasingly unable to activate NO synthesis, but all caused some activation of cytochrome c reduction compared with CaM-free nNOS. The magnitude by which each chimera activated NO synthesis was approximately proportional to the rate of heme iron reduction supported by each chimera, which varied from 0% to approximately 80% compared with native CaM and remained coupled to NO synthesis in all cases. In contrast, chimera activation of cytochrome c reduction was not always associated with accelerated reduction of nNOS flavins, and certain chimeras activated cytochrome c reduction without triggering heme iron reduction. We conclude: 1) CaM effects on electron transfer at two points within nNOS can be functionally separated. 2) CaM controls NO synthesis by governing heme iron reduction, but enhances reductase activity by two mechanisms, only one of which is associated with an increased rate of flavin reduction.
Subject(s)
Neurons/enzymology , Nitric Oxide Synthase/metabolism , Recombinant Fusion Proteins/chemistry , Amino Acid Sequence , Animals , Brain/physiology , Calmodulin/chemistry , Calmodulin/pharmacology , Cytochrome c Group/metabolism , Electron Transport/physiology , Enzyme Activation/physiology , Flavoproteins/metabolism , Heme/metabolism , Kinetics , Molecular Sequence Data , NADP/metabolism , Nitric Oxide/metabolism , Rats , Recombinant Fusion Proteins/pharmacology , Sequence Alignment , Troponin C/chemistry , Troponin C/pharmacologyABSTRACT
The nitric oxide synthase oxygenase domain (NOSox) oxidizes arginine to synthesize the cellular signal and defensive cytotoxin nitric oxide (NO). Crystal structures determined for cytokine-inducible NOSox reveal an unusual fold and heme environment for stabilization of activated oxygen intermediates key for catalysis. A winged beta sheet engenders a curved alpha-beta domain resembling a baseball catcher's mitt with heme clasped in the palm. The location of exposed hydrophobic residues and the results of mutational analysis place the dimer interface adjacent to the heme-binding pocket. Juxtaposed hydrophobic O2- and polar L-arginine-binding sites occupied by imidazole and aminoguanidine, respectively, provide a template for designing dual-function inhibitors and imply substrate-assisted catalysis.
Subject(s)
Caenorhabditis elegans Proteins , Homeodomain Proteins/genetics , Isoenzymes/chemistry , Nitric Oxide Synthase/chemistry , Protein Conformation , Amino Acid Sequence , Arginine/chemistry , Arginine/metabolism , Binding Sites , Biopterins/analogs & derivatives , Biopterins/metabolism , Catalysis , Crystallography, X-Ray , Dimerization , Enzyme Induction , Enzyme Inhibitors/metabolism , Guanidines/metabolism , Heme/chemistry , Homeodomain Proteins/chemistry , Homeodomain Proteins/physiology , Hydrogen Bonding , Imidazoles/metabolism , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Models, Molecular , Molecular Sequence Data , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/metabolism , Oxidation-Reduction , Oxygen/metabolism , Oxygenases/chemistry , Oxygenases/metabolism , Protein Folding , Protein Structure, SecondaryABSTRACT
Nitric oxide synthases (NOS) are hemeproteins that catalyze oxidation of L-arginine to nitric oxide (NO) and citrulline. The NOS heme iron is expected to participate in oxygen activation during catalysis, but its interactions with O2 are not characterized. We utilized the heme-containing oxygenase domain of neuronal NOS (nNOSoxy) and stopped-flow methods to study formation and autooxidative decomposition of the nNOSoxy oxygenated complex at 10 degrees C. Mixing ferrous nNOSoxy with air-saturated buffer generated a transient species with absorption maxima at 427 and approximately 560 nm. This species decayed within 1 s to form ferric nNOSoxy. Its formation was first order with respect to O2, monophasic, and gave rate constants for kon = 9 x 10(5) M-1 s-1 and koff = 108 s-1 for an L-arginine- and tetrahydrobiopterin (H4B)-saturated nNOSoxy. Omission of L-arginine and/or H4B did not greatly effect O2 binding and dissociation rates. Decomposition of the oxygenated intermediate was independent of O2 concentration and was either biphasic or monophasic depending on sample conditions. L-Arginine stabilized the oxygenated intermediate (decay rate = 0.14 s-1), while H4B accelerated its decay by a factor of 70 irrespective of L-arginine. The spectral and kinetic properties of the intermediate identify it as the FeIIO2 complex of nNOSoxy. Destabilization of a metallo-oxy species by H4B is unprecedented and may be important regarding the role of this cofactor in NO synthesis.
Subject(s)
Arginine/pharmacology , Biopterins/analogs & derivatives , Ferrous Compounds/metabolism , Neurons/enzymology , Nitric Oxide Synthase/metabolism , Oxygen/metabolism , Animals , Biopterins/pharmacology , Catalysis , Enzyme Stability , Heme/metabolism , Models, Chemical , Rats , Spectrophotometry, AtomicABSTRACT
The oxygenase domain of the mouse cytokine-inducible nitric-oxide synthase (iNOSox, amino acids 1-498) binds heme, tetrahydrobiopterin, and the substrate Arg and is the domain responsible for catalyzing nitric oxide synthesis and maintaining the enzyme's active dimeric structure. To further understand iNOSox structure-function, we carried out alanine point mutagenesis on 15 conserved acidic residues located within a region of iNOSox (amino acids 352-473) that shares sequence homology with the pterin-binding module in dihydrofolate reductases and may be important for iNOSox subunit dimerization and/or Arg binding. Five point mutants were identical or nearly identical to wild-type, while 10 exhibited a range of defects that included low heme content (2), heme ligand instability (2), defective dimerization (2), and poor Arg and/or tetrahydrobiopterin binding (4). Mutations that caused defective tetrahydrobiopterin binding were also associated with other defects. In contrast, two mutants (E371A and D376A) exhibited an exclusive defect in Arg binding. These mutants were dimeric, indicating that dimerization of iNOSox in Escherichia coli does not require Arg. In one case (E371A), the defect in Arg binding was absolute, as assessed by spectral perturbation, radioligand binding, and catalytic studies. We conclude that mutagenesis of conserved acidic residues within this region of iNOSox can lead to exclusive defects in dimerization and in Arg binding. Modeling considerations predict that the E371 carboxylate may participate in Arg binding by interacting with its guanidine moiety.
Subject(s)
Arginine/metabolism , Glutamic Acid/metabolism , Nitric Oxide Synthase/metabolism , Alanine , Amino Acid Sequence , Animals , Antioxidants/metabolism , Biopterins/analogs & derivatives , Biopterins/metabolism , Cattle , Chromatography, Gel , Heme/metabolism , Humans , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Nitric Oxide Synthase/genetics , Oxygenases/genetics , Oxygenases/metabolism , Rats , Structure-Activity RelationshipABSTRACT
Flavobacterium rigense strain PR2, a broad-spectrum mercury-resistant bacterium abundantly present in soil exhibited multiple metal resistance properties. Mercury resistance was due to the sequential action of two mercury-detoxicating enzymes, organomercurial lyase and mercuric reductase. The levels of these enzyme activities were determined using different mercury compounds as inducers and substrates. Mercuric reductase was partially purified from the bacterium and the physicochemical properties of the enzyme were studied. The effect of several enzyme inhibitors and heavy metal ions on the enzyme activity was also studied.
Subject(s)
Bacterial Proteins/metabolism , Flavobacterium/enzymology , Lyases/metabolism , Mercury/pharmacology , Organomercury Compounds/pharmacology , Oxidoreductases/metabolism , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/isolation & purification , Drug Resistance, Microbial , Enzyme Inhibitors/pharmacology , Flavobacterium/drug effects , Flavobacterium/isolation & purification , Organomercury Compounds/metabolism , Oxidoreductases/antagonists & inhibitors , Oxidoreductases/isolation & purification , Pesticide Residues/metabolism , Soil Microbiology , Soil Pollutants/metabolismABSTRACT
Rat neuronal NO synthase (nNOS) is comprised of a flavin-containing reductase domain and a heme-containing oxygenase domain. Calmodulin binding to nNOS increases the rate of electron transfer from NADPH into its flavins, triggers electron transfer from flavins to the heme, activates NO synthesis, and increases reduction of artificial electron acceptors such as cytochrome c. To investigate what role the reductase domain plays in calmodulin's activation of these functions, we overexpressed a form of the nNOS reductase domain (amino acids 724-1429) in the yeast Pichia pastoris that for the first time exhibits a complete calmodulin response. The reductase domain was purified by 2',5'-ADP affinity chromatography yielding 25 mg of pure protein per liter of culture. It contained 1 FAD and 0.8 FMN per molecule. Most of the protein as isolated contained an air-stable flavin semiquinone radical that was sensitive to FeCN6 oxidation. Anaerobic titration of the FeCN6-oxidized reductase domain with NADPH indicated the flavin semiquinone re-formed after addition of 1-electron equivalent and the flavins could accept up to 3 electrons from NADPH. Calmodulin binding to the recombinant reductase protein increased its rate of NADPH-dependent flavin reduction and its rate of electron transfer to cytochrome c, FeCN6, or dichlorophenolindophenol to fully match the rate increases achieved when calmodulin bound to native full-length nNOS. Calmodulin's activation of the reductase protein was associated with an increase in domain tryptophan and flavin fluorescence. We conclude that many of calmodulin's actions on native nNOS can be fully accounted for through its interaction with the nNOS reductase domain itself.