The hydrogen-bonding network in heme oxygenase also functions as a modulator of enzyme dynamics: chaotic motions upon disrupting the H-bond network in heme oxygenase from Pseudomonas aeruginosa

Relaxation compensated Carr-Purcell-Meiboom-Gill (rc-CPMG) NMR experiments have been used to investigate micros-ms motions in heme oxygenase from Pseudomonas aeruginosa (pa-HO) in its ferric state, inhibited by CN- (pa-HO-CN) and N3- (pa-HO-N3), and in its ferrous state, inhibited by CO (pa-HO-CO). Comparative analysis of the data from the three forms indicates that the nature of the coordinated distal ligand affects the micros-ms conformational freedom of the polypeptide in regions of the enzyme far removed from the heme iron and distal ligand. Interpretation of the dynamical information in the context of the crystal structure of resting state pa-HO shows that residues involved in the network of structural hydrogen-bonded waters characteristic of HOs undergo micros-ms motions in pa-HO-CN, which was studied as a model of the highly paramagnetic S = 5/2 resting state form. In comparison, similar motions are suppressed in the pa-HO-CO and pa-HO-N3 complexes, which were studied as mimics of the obligatory oxyferrous and ferric hydroperoxide intermediates, respectively, in the catalytic cycle of heme degradation. These findings suggest that in addition to proton delivery to the nascent Fe(III)-OO(-) intermediate during catalysis, the hydrogen-bonding network serves two additional roles: (i) propagate the electronic state (reactive state) in each of the distinct steps of the catalytic cycle to key but remote sections of the polypeptide via small rearrangements in the network of hydrogen bonds and (ii) modulate the conformational freedom of the enzyme, thus allowing it to adapt to the demanding changes in axial coordination state and substrate transformations that take place during the catalytic cycle. This idea was probed by disrupting the hydrogen-bonding network in pa-HO by replacing R80 with L. NMR spectroscopic studies conducted with R80L-pa-HO-N3 and R80L-pa-HO-CO revealed that the mutant exhibits nearly global conformational disorder, which is absent in the equivalent complexes of the wild type enzyme. The "chaotic" disorder in the R80L mutant is likely related to its significantly lower efficiency to hydroxylate heme in the presence of H2O2, relative to the wild type enzyme.     

The hydroxide complex of Pseudomonas aeruginosa heme oxygenase as a model of the low-spin iron(III) hydroperoxide intermediate in heme catabolism: 13C NMR spectroscopic studies suggest the active participation of the heme in macrocycle hydroxylation

13C NMR spectroscopic studies have been conducted with the hydroxide complex of Pseudomonas aeruginosa heme oxygenase (Fe(III)-OH), where OH(-) has been used as a model of the OOH(-) ligand to gain insights regarding the elusive ferric hydroperoxide (Fe(III)-OOH) intermediate in heme catabolism at ambient temperatures. Analysis of the heme core carbon resonances revealed that the coordination of hydroxide in the distal site of the enzyme results in the formation of at least three populations of Fe(III)-OH complexes with distinct electronic configurations and nonplanar ring distortions that are in slow exchange relative to the NMR time scale. The most abundant population exhibits a spin crossover between S = (1)/(2) and S = (3)/(2) spin states, and the two less abundant populations exhibit pure, S = (3)/(2) and S = (1)/(2), (d(xy)())(1) electronic configurations. We propose that the highly organized network of water molecules in the distal pocket of heme oxygenase, by virtue of donating a hydrogen bond to the coordinated hydroxide ligand, lowers its ligand field strength, thereby increasing the field strength of the porphyrin (equatorial) ligand, which results in nonplanar deformations of the macrocycle. This tendency to deform from planarity, which is imparted by the ligand field strength of the coordinated OH(-), is likely reinforced by the flexibility of the distal pocket in HO. These findings suggest that if the ligand field strength of the coordinated OOH(-) in heme oxygenase is modulated in a similar manner, the resultant large spin density at the meso carbons and nonplanar deformations of the pophyrin ring prime the macrocycle to actively participate in its own hydroxylation.     

Siderophore as a Potential Plant Growth-Promoting Agent Produced by Pseudomonas aeruginosa JAS-25

Siderophores scavenges Fe⁺³ from the vicinity of the roots of plants, and thus limit the amount of iron required for the growth of pathogens such as Fusarium oxysporum, Pythium ultimum, and Fusarium udum, which cause wilt and root rot disease in crops. The ability of Pseudomonas to grow and to produce siderophore depends upon the iron content, pH, and temperature. Maximum yield of siderophore of 130 μM was observed at pH 7.0?±?0.2 and temperature of 30 °C at 30 h. The threshold level of iron was 50 μM, which increases up to 150 μM, favoring growth but drastically affecting the production of siderophore by Pseudomonas aeruginosa JAS-25. The seeds of agricultural crops like Cicer arietinum (chick pea), Cajanus cajan (pigeon pea), and Arachis hypogaea (ground nut) were treated with P. aeruginosa JAS-25, which enhanced the seed germination, root length, shoot length, and dry weight of chick pea, pigeon pea, and ground nut plants under pot studies. The efficient growth of the plants was not only due to the biocontrol activity of the siderophore produced by P. aeruginosa JAS-25 but also may be by the production of indole acetic acid (IAA), which influences the growth of the plants as phytohormones.     

Alkyl peroxides reveal the ring opening mechanism of verdoheme catalyzed by heme oxygenase

Heme oxygenase (HO) catalyzes heme catabolism through three successive oxygenation steps where the substrate heme itself activates O2. Although a rate-determining step of the HO catalysis is considered as third oxygenation, the verdoheme degradation mechanism has been the least understood in the HO catalysis. In order to discriminate three possible pathways proposed for the verdoheme ring-opening, we have examined reactions of the verdoheme-HO-1 complex with alkyl peroxides, namely MeOOH. Under reducing conditions, the MeOOH reaction afforded two novel products whose absorption spectra are similar to but slightly different from that of biliverdin. HPLC, ESI-MS, and NMR analysis show that these products are 1- and 19-methoxy-deoxy-biliverdins. The addition of a methoxy group at one end of the linear tetrapyrrole unambiguously indicates transient formation of the Fe-OOMe intermediate and rearrangement of its terminal methoxy group to the alpha-pyrrole carbon. The corresponding OH transfer of the Fe-OOH species is highly probable in the H2O2-dependent verdoheme degradation and is likely to be the case in the O2-dependent reaction catalyzed by HO as well.     

Short-chain reactive probes as tools to unravel the Pseudomonas aeruginosa quorum sensing regulon

In recent years, the world has seen a troubling increase in antibiotic resistance among bacterial pathogens. In order to provide alternative strategies to combat bacterial infections, it is crucial deepen our understanding into the mechanisms that pathogens use to thrive in complex environments. Most bacteria use sophisticated chemical communication systems to sense their population density and coordinate gene expression in a collective manner, a process that is termed “quorum sensing” (QS). The human pathogen Pseudomonas aeruginosa uses several small molecules to regulate QS, and one of them is N-butyryl-l-homoserine lactone (C₄-HSL). Using an activity-based protein profiling (ABPP) strategy, we designed biomimetic probes with a photoreactive group and a ‘click’ tag as an analytical handle. Using these probes, we have identified previously uncharacterized proteins that are part of the P. aeruginosa QS network, and we uncovered an additional role for this natural autoinducer in the virulence regulon of P. aeruginosa, through its interaction with PhzB1/2 that results in inhibition of pyocyanin production.

Short-chain reactive probes can be used as tools to shed new light on virulence mechanisms in bacterial pathogens.     

Conversion of fatty aldehydes to alka(e)nes and formate by a cyanobacterial aldehyde decarbonylase: cryptic redox by an unusual dimetal oxygenase

Cyanobacterial aldehyde decarbonylase (AD) catalyzes conversion of fatty aldehydes (R-CHO) to alka(e)nes (R-H) and formate. Curiously, although this reaction appears to be redox-neutral and formally hydrolytic, AD has a ferritin-like protein architecture and a carboxylate-bridged dimetal cofactor that are both structurally similar to those found in di-iron oxidases and oxygenases. In addition, the in vitro activity of the AD from Nostoc punctiforme (Np) was shown to require a reducing system similar to the systems employed by these O(2)-utilizing di-iron enzymes. Here, we resolve this conundrum by showing that aldehyde cleavage by the Np AD also requires dioxygen and results in incorporation of (18)O from (18)O(2) into the formate product. AD thus oxygenates, without oxidizing, its substrate. We posit that (i) O(2) adds to the reduced cofactor to generate a metal-bound peroxide nucleophile that attacks the substrate carbonyl and initiates a radical scission of the C1-C2 bond, and (ii) the reducing system delivers two electrons during aldehyde cleavage, ensuring a redox-neutral outcome, and two additional electrons to return an oxidized form of the cofactor back to the reduced, O(2)-reactive form.     

Theory favors a stepwise mechanism of porphyrin degradation by a ferric hydroperoxide model of the active species of heme oxygenase

The report uses density functional theory to address the mechanism of heme degradation by the enzyme heme oxygenase (HO) using a model ferric hydroperoxide complex. HO is known to trap heme molecules and degrade them to maintain iron homeostasis in the biosystem. The degradation is initiated by complexation of the heme, then formation of the iron-hydroperoxo species, which subsequently oxidizes the meso position of the porphyrin by hydroxylation, thereby enabling eventually the cleavage of the porphyrin ring. Kinetic isotope effect studies indicate that the mechanism is assisted by general acid catalysis, via a chain of water molecules, and that all the events occur in concert. However, previous theoretical treatments indicated that the concerted mechanism has a high barrier, much higher than an alternative mechanism that is initiated by O-O bond homolysis of iron-hydroperoxide. The present contribution studies the stepwise and concerted acid-catalyzed mechanisms using H(3)O(+)(H(2)O)(n)(), n = 0-2. The effect of the acid strength is tested using the H(4)N(+)(H(2)O)(2) cluster and a fully protonated ferric hydroperoxide. All the calculations show that a stepwise mechanism that involves proton relay and O-O homolysis, in the rate-determining step, has a much lower barrier (>10 kcal/mol) than the corresponding fully concerted mechanism. The best fit of the calculated solvent kinetic isotope effect, to the experimental data, is obtained for the H(3)O(+)(H(2)O)(2) cluster. The calculated alpha-deuterium secondary kinetic isotope effect is inverse (0.95-0.98), but much less so than the experimental value (0.7). Possible reasons for this quantitative difference are discussed. Some probes are suggested that may enable experiment to distinguish the stepwise from the concerted mechanism.     

Permeabilization of biological and artificial membranes by a bacterial dirhamnolipid produced by Pseudomonas aeruginosa

Pseudomonas aeruginosa, when cultured under the appropriate conditions, secretes rhamnolipids to the external medium. These glycolipids constitute one of the most interesting classes of biosurfactants so far. A dirhamnolipid fraction was isolated and purified from the crude biosurfactant, and its action on model and biological membranes was studied. Dirhamnolipid induced leakage of internal contents, as measured by the release of carboxyfluorescein, in phosphatidylcholine unilamellar vesicles, at concentrations below its CMC. Membrane solubilization was not observed within this concentration range. The presence of inverted cone-shaped lipids in the membrane, namely lysophosphatidylcholine, accelerated leakage, whereas cone-shaped lipids, like phosphatidylethanolamine, decreased leakage rate. Increasing concentrations of cholesterol protected the membrane against dirhamnolipid-induced leakage, which was totally abolished by the presence of 50 mol% of the sterol. Dirhamnolipid caused hemolysis of human erythrocytes through a lytic mechanism, as shown by the similar rates of K⁺ and hemoglobin leakage, and by the absence of effect of osmotic protectants. Scanning electron microscopy showed that the addition of the biosurfactant changed the usual disc shape of erythrocytes into that of spheroechinocytes. The results are discussed within the frame of the biological actions of dirhamnolipid, and the possible future applications of this biosurfactant.     

Quantum mechanical/molecular mechanical study of mechanisms of heme degradation by the enzyme heme oxygenase: the strategic function of the water cluster

Heme degradation by heme oxygenase (HO) enzymes is important in maintaining iron homeostasis and prevention of oxidative stress, etc. In response to mechanistic uncertainties, we performed quantum mechanical/molecular mechanical investigations of the heme hydroxylation by HO, in the native route and with the oxygen surrogate donor H2O2. It is demonstrated that H2O2 cannot be deprotonated to yield Fe(III)OOH, and hence the surrogate reaction starts from the FeHOOH complex. The calculations show that, when starting from either Fe(III)OOH or Fe(III)HOOH, the fully concerted mechanism involving O-O bond breakage and O-C(meso) bond formation is highly disfavored. The low-energy mechanism involves a nonsynchronous, effectively concerted pathway, in which the active species undergoes first O-O bond homolysis followed by a barrier-free (small with Fe(III)HOOH) hydroxyl radical attack on the meso position of the porphyrin. During the reaction of Fe(III)HOOH, formation of the Por+*FeIV=O species, compound I, competes with heme hydroxylation, thereby reducing the efficiency of the surrogate route. All these conclusions are in accord with experimental findings (Chu, G. C.; Katakura, K.; Zhang, X.; Yoshida, T.; Ikeda-Saito, M. J. Biol. Chem. 1999, 274, 21319). The study highlights the role of the water cluster in the distal pocket in creating "function" for the enzyme; this cluster affects the O-O cleavage and the O-Cmeso formation, but more so it is responsible for the orientation of the hydroxyl radical and for the observed alpha-meso regioselectivity of hydroxylation (Ortiz de Montellano, P. R. Acc. Chem. Res. 1998, 31, 543). Differences/similarities with P450 and HRP are discussed.     

How heme metabolism occurs in heme oxygenase: computational study of oxygen-donation ability of the oxo and hydroperoxo species

We report a density functional theory study on the heme metabolism in heme oxygenase using iron-hydroperoxo and -oxo models. The activation energies for heme oxidation at the alpha-carbon by the iron-hydroperoxo and -oxo species are calculated to be 42.9 and 39.9 kcal/mol, respectively. These high activation barriers lead us to reconsider the catalytic mechanism of heme oxygenase     

Enhancement of rhamnoplipid production in residual soybean oil by an isolated strain of Pseudomonas aeruginosa

In the present work, the production of rhamnolipid from residual soybean oil (RSO) from food frying facilities was studied using a strain of Pseudomonas aeruginosa of contaminated lagoon, isolated from a hydrocarbon contaminated soil. The optimization of RSO, ammonium nitrate, and brewery residual yeast concentrations was accomplished by a central composite experimental design and surface response analysis. The experiments were performed in 500-mL Erlenmeyer flasks containing 50 mL of mineral medium, at 170 rpm and 30 +/- 1 degrees C, for a 48-h fermentation period. Rhamnolipid production has been monitored by measurements of surface tension, rhamnose concentration, and emulsifying activity. The best-planned results, located on the central point, have corresponded to 22 g/L of RSO, 5.625 g/L of NH(4)NO(3), and 11.5 g/L of brewery yeast. At the maximum point the values for rhamnose and emulsifying index were 2.2 g/L and 100%, respectively.     

Comparative effects of curcuminoids on endothelial heme oxygenase-1 expression: ortho-methoxy groups are essential to enhance heme oxygenase activity and protection

Recently, it has been reported that curcumin, which is known as a potent antioxidant, acts as a non- stressful and non-cytotoxic inducer of the cytoprotective heme oxygenase (HO)-1. In this study, naturally occurring curcuminoids, such as pure curcumin, demethoxycurcumin (DMC) and bis-demethoxycurcumin (BDMC), were compared for their potential ability to modulate HO-1 expression and cytoprotective activity in human endothelial cells. All three curcuminoids could induce HO-1 expression and HO activity with differential levels. The rank order of HO activity was curcumin, DMC and BDMC. In comparison with endothelial protection against H2O2-induced cellular injury, cytoprotective capacity was found to be highest with curcumin, followed by DMC and BDMC. Interestingly, cytoprotective effects afforded by curcuminoids were considerably associated with their abilities to enhance HO activity. Considering that the main difference among the three curcuminoids is the number of methoxy groups (none for BDMC, one for DMC, and two for curcumin), the presence of methoxy groups in the ortho position on the aromatic ring was suggested to be essential to enhance HO-1 expression and cytoprotection in human endothelial cells. Our results may be useful in designing more efficacious HO-1 inducers which could be considered as promising pharmacological agents in the development of therapeutic approaches for the prevention or treatment of endothelial diseases caused by oxidative damages.     

Silver nanoparticles impede the biofilm formation by Pseudomonas aeruginosa and Staphylococcus epidermidis

Biofilms are ensued due to bacteria that attach to surfaces and aggregate in a hydrated polymeric matrix. Formation of these sessile communities and their inherent resistance to anti-microbial agents are the source of many relentless and chronic bacterial infections. Such biofilms are responsible play a major role in development of ocular related infectious diseases in human namely microbial keratitis. Different approaches have been used for preventing biofilm related infections in health care settings. Many of these methods have their own demerits that include chemical based complications; emergent antibiotic resistant strains, etc. silver nanoparticles are renowned for their influential anti-microbial activity. Hence the present study over the biologically synthesized silver nanoparticles, exhibited a potential anti-biofilm activity that was tested in vitro on biofilms formed by Pseudomonas aeruginosa and Staphylococcus epidermidis during 24-h treatment. Treating these organisms with silver nanoparticles resulted in more than 95% inhibition in biofilm formation. The inhibition was known to be invariable of the species tested. As a result this study demonstrates the futuristic application of silver nanoparticles in treating microbial keratitis based on its potential anti-biofilm activity.     

Thermodynamics of the interaction of a dirhamnolipid biosurfactant secreted by Pseudomonas aeruginosa with phospholipid membranes

Rhamnolipids are bacterial biosurfactants produced by Pseudomonas spp. These compounds have been shown to present several interesting biological activities, restricting the growth of Bacillus subtilis and showing zoosporicidal activity on zoosporic phytopathogens. It has been suggested that the interaction with the membrane could ultimately be responsible for these actions. Therefore, it is of great interest to gain insight into the molecular mechanism of the interaction of purified rhamnolipids with the various phospholipid components of biological membranes. In this work, the critical micelle concentration (cmc) of a purified dirhamnolipid produced by Pseudomonas aeruginosa has been determined both by isothermal titration calorimetry and surface tension measurements. The partition coefficients from water to membranes of different compositions, as well as the corresponding thermodynamic parameters, have been determined by isothermal titration calorimetry measurements. The results indicate that dirhamnolipid membrane partitioning is an entropically driven process. The presence of cholesterol in the membrane decreases the partition of dirhamnolipid. On the other hand, phosphatidylethanolamine stimulates dirhamnolipid binding, whereas lysophosphatidylcholine opposes binding, suggesting that the biosurfactant behaves as an inverted-cone-shaped molecule. The values obtained for the cmc and the partition constant are considered in relation to the surfactant potency of dirhamnolipids.     

Aggregation behaviour of a dirhamnolipid biosurfactant secreted by Pseudomonas aeruginosa in aqueous media

The process of micelle formation, along with the formation of higher order aggregates, is described for a dirhamnolipid extracellular biosurfactant secreted by Pseudomonas aeruginosa. As determined by surface tension measurements, at pH 7.4 the CMC of dirhamnolipid is 0.110 mM, whereas at pH 4.0 it falls to 0.010 mM, indicating that the negatively charged diRL has a much higher CMC value than the neutral species. Centrifugation and dynamic light scattering measurements show formation of larger aggregates at concentrations above the CMC. These aggregates have been shown by electron microscopy to be mainly multilamellar vesicles of heterogeneous size. X-ray scattering gave a value of 32 A for the interlamellar repeat distance of these vesicles. Taking into account the experimental data, a molecular modelling of the dirhamnolipid moiety has been carried out, which details the size of the hydrophilic and hydrophobic portions, and suggests the possible intermolecular interactions responsible for the stabilisation of dirhamnolipid aggregates. The relevance of this aggregation behaviour is discussed with respect to the molecular basis of its activities.     

Siderophore production by a marine Pseudomonas aeruginosa and its antagonistic action against phytopathogenic fungi

A marine isolate of fluorescent Pseudomonas sp. having the ability to produce the pyoverdine type of siderophores under low iron stress (up to 10 μM iron in the succinate medium) was identified as Pseudomonas aeruginosa by using BIOLOG Breathprint and siderotyping. Pyoverdine production was optimum at 0.2% (w/v) succinate, pH 6.0, in an iron-deficient medium. Studies carried out in vitro revealed that purified siderophores and Pseudomonas culture have good antifungal activity against the plant deleterious fungi, namely, Aspergillus niger, Aspergillus flavus, Aspergillus oryzae, Fusarium oxysporum, and Sclerotium rolfsii. Siderophore-based maximum inhibition was observed against A. niger. These in vitro antagonistic actions of marine Pseudomonas against phytopathogens suggest the potential of the organism to serve as a biocontrol agent.     

氧气催化氧化环己烷

本文系统综述了O₂氧化剂用于环己烷催化氧化体系的研究进展,包括金属配合物催化、金属纳米粒子催化、金属氧化物粒子催化、分子筛催化、碳材料催化、光促进催化、杂多酸催化、金属-有机骨架材料催化等。本文认为研究、开发以O₂为氧化剂,高活性高选择性的非均相环己烷催化氧化体系将成为今后环己烷催化氧化研究的主要方向,尤其是多金属甚至多元素复合体系。本综述不仅对开发高催化活性高选择性的环己烷催化氧化体系,改进目前工业上的环己醇环己酮制备工艺具有重要的参考价值,而且还对其他烃类C-H键和C-C键高效催化氧化体系甚至其他氧化体系的研究与开发也具有重要的参考价值。     

GC-MS Analysis and Microbiological Evaluation of Caraway Essential Oil as a Virulence Attenuating Agent against Pseudomonas aeruginosa

The emergence of resistant microbes threatens public health on our planet, and the emergence of resistant bacteria against the most commonly used antibiotics necessitates urgent alternative therapeutic options. One way to fight resistant microbes is to design new antimicrobial agents, however, this approach takes decades of research. An alternative or parallel approach is to target the virulence of bacteria with natural or synthetic agents. Active constituents from medicinal plants represent a wide library to screen for natural anti-virulence agents. Caraway is used as a traditional spice and in some medicinal applications such as carminative, antispasmodic, appetizer, and expectorant. Caraway essential oil is rich in terpenes that were previously reported to have antimicrobial activities. In our study, we tested the caraway essential oil in sub-inhibitory concentration as a virulence agent against the Gram-negative bacteria Pseudomonas aeruginosa. Caraway essential oil in sub-inhibitory concentration dramatically blocked protease activity, pyocyanin production, biofilm formation, and quorum sensing activity of P. aeruginosa. The gas chromatography–mass spectroscopy (GC-MS) profile of caraway fruit oil identified 13 compounds representing 85.4% of the total oil components with carvone and sylvestrene as the main constituents. In conclusion, caraway essential oil is a promising virulence-attenuating agent that can be used against topical infections caused by P. aeruginosa.