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The structure of the NO-sensing domain of the transcription factor DNR from Pseudomonas aeruginosa
Full text linkNitrosylation of c heme in cd(1)-nitrite reductase is enhanced during catalysis
No full textThe reduction of nitrite into nitric oxide (NO) in denitrifying bacteria is catalyzed by nitrite reductase. In several species, this enzyme is a heme-containing protein with one c heme and one d(1) heme per mono-mer (cd(1)NiR), encoded by the nirS gene.
For many years, the evidence of a link between NO and this hemeprotein represented a paradox, given that NO was known to tightly bind and, possibly, inhibit hemeproteins, including cd(1)NiRs.
It is now established that, during catalysis, cd(1)NiRs diverge from "canonical" hemeproteins, since the product NO rapidly dissociates from the ferrous d(1) heme, which, in turn, displays a peculiar "low" affinity for NO (K-D = 0.11 microM at pH 7.0).
It has been also previously shown that the c heme reacts with NO at acidic pH but c heme nitrosylation was not extensively investigated, given that in cd(1)NiR it was considered a side reaction, rather than a genuine process controlling catalysis.
The spectroscopic study of the reaction of cd(1)NiR and its semi-apo derivative (containing the sole c heme) with NO reported here shows that c heme nitrosylation is enhanced during catalysis; this evidence has been discussed in order to assess the potential of c heme nitrosylation as a regulatory process, as observed for cytochrome c nitrosylation in mammalian mitochondria
In silico discovery and in vitro validation of catechol-containing sulfonohydrazide compounds as potent inhibitors of the diguanylate cyclase PleD
No full textBiofilm formation is responsible for increased antibiotic tolerance in pathogenic bacteria. Cyclic di-GMP (c-di-GMP) is a widely used second-messenger signal, playing a key role in bacterial biofilm formation. C-di-GMP is synthesized by diguanylate cyclases (DGC), a conserved class of enzymes absent in mammals, hence considered attractive molecular targets for the development of anti-biofilm agents.Here, the results of a virtual screening approach aimed at identifying small-molecule inhibitors of the DGC PleD from Caulobacter crescentus are described. A 3D-pharmacophore model, derived from the binding mode of GTP to the active site of PleD was exploited to screen the ZINC database of compounds. Seven virtual hits were tested in vitro for their ability to inhibit the activity of purified PleD by using circular dichroism spectroscopy. Two drug-like molecules with a cathecol moiety and a sulfonohydrazide scaffold were shown to competitively inhibit PleD at low micromolar range (IC50 ≈ 11 μM). Their predicted binding mode highlighted key structural features presumably responsible for the efficient inhibition of PleD by both hits. These molecules represent the most potent in vitro inhibitors of PleD identified so far, and could therefore result in useful leads for the development of novel classes of antimicrobials able to hamper biofilm formation
The transcription factor DNR from Pseudomonas aeruginosa specifically requires nitric oxide and haem for the activation of a target promoter in Escherichia coli
No full textPseudomonas aeruginosa is a well-known pathogen in chronic respiratory diseases such as cystic fibrosis. Infectivity of P. aeruginosa is related to the ability to grow under oxygen-limited conditions using the anaerobic metabolism of denitrification, in which nitrate is reduced to dinitrogen via nitric oxide (NO). Denitrification is activated by a cascade of redox-sensitive transcription factors, among which is the DNR regulator, sensitive to nitrogen oxides. To gain further insight into the mechanism of NO-sensing by DNR we have developed an Escherichia coli-based reporter system to investigate different aspects of DNR activity. In E. coli DNR responds to NO, as shown by its ability to transactivate the P. aeruginosa norCB promoter. The direct binding of DNR to the target DNA is required, since mutations in the helix-turn-helix domain of DNR and specific nucleotide substitutions in the consensus sequence of the norCB promoter abolish the transcriptional activity. Using an E coli strain deficient in haem biosynthesis, we have also confirmed that haem is required in vivo for the NO-dependent DNR activity, in agreement with the property of DNR to bind in vitro. Finally, we have shown, we believe for the first time, that DNR is able to discriminate in vivo between different diatomic signal molecules, NO and CO, both ligands of the reduced haem iron in vitro, suggesting that DNR responds specifically to NO
An Efficient One-Pot Access to Poly(arylene ethynylene) Homopolimers: Use of the Bu3Sn-Moiety as Recyclable carrier to Introduce the Ethynyl Unit into the Chain
No full textUse of the extended one-pot (EOP) procedure for the preparation of ethynylated thiophene derivatives and related palladium-ethynylthiophene organometallic oligomers
No full textThe palladium-catalyzed coupling (Stille coupling) of 2,5-diiodothiophene (1) with tributyl(ethynyl)tin forms the 2,5-bis(ethynyl)thiophene (3) and tributyltin iodide as side product (step 1). Addition of lithium diisopropylamide (LDA) to this mixture causes deprotonation of the bis-alkyne and its reaction with the tin halide present in the medium to form the 2,5-bis[(tributyltin)ethynyl]thiophene (4) (step 2). To this mixture was subsequently added trans-dichlorobis(tri-n-butylphosphine)palladium (5), and the corresponding trans-bis(tri-n-butylphosphine)-mu- [2,5-bis(ethynyl)thiophene]palladium oligomer (6) was obtained (step 3). Alternatively, the same route can be directed toward the formation of ethynylated thiophene oligomers: after formation of the 2,5-bis[(tributyltin)ethynyl]thiophene (4) (step 2), addition of 2-iodothiophene (8) or 2-iodo-5-(trimethylsilyl)thiophene (10) led to the formation of 2,5-bis(2-thienylethynyl)thiophene (9) (step 3) and [2-trimethylsilyl(ethynyl)thiophene]-2,5-bisethynylthiophene (11) (step 3') respectively. The latter can be easily desilylated to obtain the [2-(ethynyl)thiophene]-2,5-bisethynylthiophene (13), while treatment of 9 with sec-BuLi/I-2 formed the 2,5-[2,2'-(5,5'-diiodo)bisthienyl]bisethynylthiophene (12). Through a sequence of transformations similar to steps 1-3, the oligo(iodo)ethynylthiophene 12 has been connected to the bis(tri-n-butylphosphine)palladium moiety to form the trans-bis(tri-n-butylphosphine)-,u-[2,2'-bis(ethynyl)thiophenel-2,5-bisethynylthiophene]palladium polymer (15). To compare the advantages of the above extended one-pot (EOP) procedures over classical routes, polymers 6 and 15 were also prepared by the copper-catalyzed reaction of trans-dichlorobis(tri-n-butylphosphine)palladium (5) with 2,5-bis(ethynyl)thiophene (3) and [2-(ethynyl)thiophene]-2,5-bisethynylthiophene (13)
N-oxide sensing in Pseudomonas aeruginosa: expression and preliminary characterization of DNR, an FNR-CRP type transcriptional regulator.
No full textIn denitrifying bacteria, the concentration of NO is maintained low by a tight control of the expression and activity of nitrite and NO reductases. Regulation involves redox-linked transcription factors, such as those belonging to the CRP-FNR (cAMP receptor protein-fumarate and nitrate reductase regulator) superfamily, which act as oxygen and N-oxide sensors. Given that few members of this superfamily have been characterized in detail, we have cloned, expressed and purified the dissimilative nitrate respiration regulator from Pseudomonas aeruginosa. To gain insights on the structural properties of the dissimilative nitrate respiration regulator, we have also determined the aggregation state of the purified protein and its ability to bind hydrophobic compounds such as 8-anilino-1-naphthalenesulphonic acid
N-oxide sensing and denitrification: the DNR transcription factors
No full textAll denitrifiers can keep the steady-state concentrations of nitrite and nitric oxide (NO) below cytotoxic levels by controlling the expression of denitrification gene clusters by redox signalling through transcriptional regulators belonging to the CIRP (CAMP receptor protein)/FNR (fumarate and nitrate reductase regulator) superfamily
Inhibition Of Bacterial Biofilms: New Molecular Strategies Targeting Cyclic-Di-GMP Metabolism.
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