Elucidation of the hetero-dimeric binding activity of LasR and RhlR proteins with the promoter DNA and the role of a specific Phe residue during the biosynthesis of HCN synthase from opportunistic pathogen Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen. It causes secondary infections in patients suffering from cancer and other immunological disorders. The pathogenicity of the organism is dependent on the ability of the organism to code for hydrogen cyanide (HCN), the synthesis of which is mediated by HCN synthase enzyme. HCN synthase is encoded by hcnABC operon. The transcription of the operon is controlled by a complex interplay between the proteins LasR and RhlR. Till date, there is no report that deals with the binding interactions of the RhlR-LasR heterodimer with the promoter DNA region of the hcnABC operon. We, for the first time, tried to analyse the binding modes of the RhlR-LasR heterodimer with the promoter DNA regions. From our work, we could predict the importance of a specific amino acid residue Phe214 from RhlR which might be considered to have the desired specificity to bind to the promoter DNA. Therefore, the amino acid Phe214 may be targeted to develop suitable ligands to eradicate the spread of secondary infections by Pseudomonas aeruginosa.

Decrease of Arabidopsis PAO activity entails increased RBOH activity, ROS content and altered responses to Pseudomonas.

Polyamines (PAs) are small aliphatic amines with important regulatory activities in plants. Biotic stress results in changes in PA levels due to de novo synthesis and PA oxidation. In Arabidopsis thaliana five FAD-dependent polyamine oxidase enzymes (AtPAO1-5) participate in PA back-conversion and degradation. PAO activity generates H2O2, an important molecule involved in cell signaling, elongation, programmed cell death, and defense responses. In this work we analyzed the role of AtPAO genes in the Arabidopsis thaliana-Pseudomonas syringae pathosystem. AtPAO1 and AtPAO2 genes were transcriptionally up-regulated in infected plants. Atpao1-1 and Atpao2-1 single mutant lines displayed altered responses to Pseudomonas, and an increased susceptibility was found in the double mutant Atpao1-1 x Atpao2-1. These polyamine oxidases mutant lines showed disturbed contents of ROS (H2O2 and O2-) and altered activities of RBOH, CAT and SOD enzymes both in infected and control plants. In addition, changes in the expression levels of AtRBOHD, AtRBOHF, AtPRX33, and AtPRX34 genes were also noticed. Our data indicate an important role for polyamine oxidases in plant defense and ROS homeostasis.

Mitochondrial calcium uniporter regulates PGC-1α expression to mediate metabolic reprogramming in pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with an increased mortality. Metabolic reprogramming has a critical role in multiple chronic diseases. Lung macrophages expressing the mitochondrial calcium uniporter (MCU) have a critical role in fibrotic repair, but the contribution of MCU in macrophage metabolism is not known. Here, we show that MCU regulates peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and metabolic reprogramming to fatty acid oxidation (FAO) in macrophages. MCU regulated PGC-1α expression by increasing the phosphorylation of ATF-2 by the p38 MAPK in a redox-dependent manner. The expression and activation of PGC-1α via the p38 MAPK was regulated by MCU-mediated mitochondrial calcium uptake, which is linked to increased mitochondrial ROS (mtROS) production. Mice harboring a conditional expression of dominant-negative MCU in macrophages had a marked reduction in mtROS and FAO and were protected from pulmonary fibrosis. Moreover, IPF lung macrophages had evidence of increased MCU and mitochondrial calcium, increased phosphorylation of ATF2 and p38, as well as increased expression of PGC-1α. These observations suggest that macrophage MCU-mediated metabolic reprogramming contributes to fibrotic repair after lung injury.

Theobromine and related methylxanthines as inhibitors of Primary Amine Oxidase.

Methylxanthines are among the most widely consumed drugs in the world and evidence of their health benefits has been growing in recent years. Primary Amine Oxidase (PrAO) has been recognized as a therapeutic target for the amelioration of inflammatory, vascular, and neurodegenerative diseases. Previous work in our laboratories showed that caffeine inhibited Bovine PrAO with a Ki of 1.0 mM using benzylamine as substrate. This study aimed to extend our previous work and explore the possibility that related methylxanthines might influence PrAO activity. While paraxanthine, theophylline, and 7-methylxanthine had little effect on PrAO, theobromine was a noncompetitive inhibitor with a Ki of 276 ± 44 µM. The specific structural elements of methylxanthines that are required for inhibition allow us to suggest that their binding site on PrAO may be a target for therapeutics. The health benefits associated with dietary methylxanthine consumption could involve PrAO inhibition. PRACTICAL APPLICATIONS: Inhibition of PrAO by methylxanthines may be significant in conferring health benefits. The design of PrAO inhibitors based on the structural motifs identified in this study (N-methylation at specific locations) is indicated. Existing therapeutics based on a core xanthine structure can be evaluated for their effects on PrAO. PrAO inhibition must be considered as a potential mediator of the beneficial health effects of some methylxanthines. If inhibition in human tissues is comparable to, or greater than, that found in these studies it points to an important role for these compounds in human health.

The Arabidopsis polyamine oxidase/dehydrogenase 5 interferes with cytokinin and auxin signaling pathways to control xylem differentiation.

In plants, the polyamines putrescine, spermidine, spermine (Spm), and thermospermine (Therm-Spm) participate in several physiological processes. In particular, Therm-Spm is involved in the control of xylem differentiation, having an auxin antagonizing effect. Polyamine oxidases (PAOs) are FAD-dependent enzymes involved in polyamine catabolism. In Arabidopsis, five PAOs are present, among which AtPAO5 catalyzes the back-conversion of Spm, Therm-Spm, and N1-acetyl-Spm to spermidine. In the present study, it is shown that two loss-of-function atpao5 mutants and a 35S::AtPAO5 Arabidopsis transgenic line present phenotypical differences from the wild-type plants with regard to stem and root elongation, differences that are accompanied by changes in polyamine levels and the number of xylem vessels. It is additionally shown that cytokinin treatment, which up-regulates AtPAO5 expression in roots, differentially affects protoxylem differentiation in 35S::AtPAO5, atpao5, and wild-type roots. Together with these findings, Therm-Spm biosynthetic genes, as well as auxin-, xylem-, and cytokinin-related genes (such as ACL5, SAMDC4, PIN1, PIN6, VND6, VND7, ATHB8, PHB, CNA, PXY, XTH3, XCP1, and AHP6) are shown to be differentially expressed in the various genotypes. These data suggest that AtPAO5, being involved in the control of Therm-Spm homeostasis, participates in the tightly controlled interplay between auxin and cytokinins that is necessary for proper xylem differentiation.

Mechanism of Flavoprotein l-6-Hydroxynicotine Oxidase: pH and Solvent Isotope Effects and Identification of Key Active Site Residues.

The flavoenzyme l-6-hydroxynicotine oxidase is a member of the monoamine oxidase family that catalyzes the oxidation of (S)-6-hydroxynicotine to 6-hydroxypseudooxynicotine during microbial catabolism of nicotine. While the enzyme has long been understood to catalyze oxidation of the carbon-carbon bond, it has recently been shown to catalyze oxidation of a carbon-nitrogen bond [Fitzpatrick, P. F., et al. (2016) Biochemistry 55, 697-703]. The effects of pH and mutagenesis of active site residues have now been utilized to study the mechanism and roles of active site residues. Asn166 and Tyr311 bind the substrate, while Lys287 forms a water-mediated hydrogen bond with flavin N5. The N166A and Y311F mutations result in ∼30- and ∼4-fold decreases in kcat/Km and kred for (S)-6-hydroxynicotine, respectively, with larger effects on the kcat/Km value for (S)-6-hydroxynornicotine. The K287M mutation results in ∼10-fold decreases in these parameters and a 6000-fold decrease in the kcat/Km value for oxygen. The shapes of the pH profiles are not altered by the N166A and Y311F mutations. There is no solvent isotope effect on the kcat/Km value for amines. The results are consistent with a model in which both the charged and neutral forms of the amine can bind, with the former rapidly losing a proton to a hydrogen bond network of water and amino acids in the active site prior to the transfer of hydride to the flavin.

Polyamine oxidase 5 loss-of-function mutations in Arabidopsis thaliana trigger metabolic and transcriptional reprogramming and promote salt stress tolerance.

The family of polyamine oxidases (PAO) in Arabidopsis (AtPAO1-5) mediates polyamine (PA) back-conversion, which reverses the PA biosynthetic pathway from spermine and its structural isomer thermospermine (tSpm) into spermidine and then putrescine. Here, we have studied the involvement of PA back-conversion in Arabidopsis salinity tolerance. AtPAO5 is the Arabidopsis PAO gene member most transcriptionally induced by salt stress. Two independent loss-of-function mutants (atpao5-2 and atpao5-3) were found to exhibit constitutively higher tSpm levels, with associated increased salt tolerance. Using global transcriptional and metabolomic analyses, the underlying mechanisms were studied. Stimulation of abscisic acid and jasmonate (JA) biosynthesis and accumulation of important compatible solutes, such as sugars, polyols and proline, as well as TCA cycle intermediates were observed in atpao5 mutants under salt stress. Expression analyses indicate that tSpm modulates the transcript levels of several target genes, including many involved in the biosynthesis and signalling of JA, some of which are already known to promote salinity tolerance. Transcriptional modulation by tSpm is isomer-dependent, thus demonstrating the specificity of this response. Overall, we conclude that tSpm triggers metabolic and transcriptional reprogramming that promotes salt stress tolerance in Arabidopsis.

Proteomic analysis of cow, yak, buffalo, goat and camel milk whey proteins: quantitative differential expression patterns.

To aid in unraveling diverse genetic and biological unknowns, a proteomic approach was used to analyze the whey proteome in cow, yak, buffalo, goat, and camel milk based on the isobaric tag for relative and absolute quantification (iTRAQ) techniques. This analysis is the first to produce proteomic data for the milk from the above-mentioned animal species: 211 proteins have been identified and 113 proteins have been categorized according to molecular function, cellular components, and biological processes based on gene ontology annotation. The results of principal component analysis showed significant differences in proteomic patterns among goat, camel, cow, buffalo, and yak milk. Furthermore, 177 differentially expressed proteins were submitted to advanced hierarchical clustering. The resulting clustering pattern included three major sample clusters: (1) cow, buffalo, and yak milk; (2) goat, cow, buffalo, and yak milk; and (3) camel milk. Certain proteins were chosen as characterization traits for a given species: whey acidic protein and quinone oxidoreductase for camel milk, biglycan for goat milk, uncharacterized protein (Accession Number: F1MK50 ) for yak milk, clusterin for buffalo milk, and primary amine oxidase for cow milk. These results help reveal the quantitative milk whey proteome pattern for analyzed species. This provides information for evaluating adulteration of specific specie milk and may provide potential directions for application of specific milk protein production based on physiological differences among animal species.

From caffeine to fish waste: amine compounds present in food and drugs and their interactions with primary amine oxidase.

Tissue bound primary amine oxidase (PrAO) and its circulating plasma-soluble form are involved, through their catalytic activity, in important cellular roles, including the adhesion of lymphocytes to endothelial cells during various inflammatory conditions, the regulation of cell growth and maturation, extracellular matrix deposition and maturation and glucose transport. PrAO catalyses the oxidative deamination of several xenobiotics and has been linked to vascular toxicity, due to the generation of cytotoxic aldehydes. In this study, a series of amines and aldehydes contained in food and drugs were tested via a high-throughput assay as potential substrates or inhibitors of bovine plasma PrAO. Although none of the compounds analyzed were found to be substrates for the enzyme, a series of molecules, including caffeine, the antidiabetics phenformin and tolbutamide and the antimicrobial pentamidine, were identified as PrAO inhibitors. Although the inhibition observed was in the millimolar and micromolar range, these data show that further work will be necessary to elucidate whether the interaction of ingested biogenic or xenobiotic amines with PrAO might adversely affect its biological roles.

The human lysyl oxidase-like 2 protein functions as an amine oxidase toward collagen and elastin.

The lysyl oxidase-like 2 (LOXL2) protein is a human paralogue of lysyl oxidase (LOX) that functions as an amine oxidase for formation of lysine-derived cross-links found in collagen and elastin. In addition to the C-terminal domains characteristic to the LOX family members, LOXL2 contains four scavenger receptor cysteine-rich (SRCR) domains in the N-terminus. In order to assess the amine oxidase activity of LOXL2, we expressed a series of recombinant LOXL2 proteins with deletions in the SRCR domains, using an Escherichia coli expression system. All of the purified recombinant LOXL2 proteins, with or without the SRCR domains in the N-terminus, showed significant amine oxidase activity toward several different types of collagen and elastin in in vitro amine oxidase assays, indicating deletion of the SRCR domains does not interfere with amine oxidase activity of LOXL2. Further, amine oxidase activity of LOXL2 was not susceptible to inhibition by ß-aminopropionitrile, an irreversible inhibitor of LOX, suggesting a different enzymatic mechanism between these two paralogues.

Antioxidant and amine oxidase inhibitory activities of hydroxyurea.

Hydroxyurea (HU, NH(2)CONHOH), or hydroxycarbamide, is a hydroxamic acid derivative used as a drug for anti-neoplasm and sickle-cell disease. In this study, HU was found to have antioxidant activities against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radicals and dose-dependent inhibitory activities against monoamine oxidase (MAO)-A, MAO-B, and semicarbazide-sensitive amine oxidase (SSAO) as compared to controls of clorgyline, deprenyl, and semicarbazide respectively. HU showed mixed-type, competitive-type, and competitive-type inhibition, respectively, with respect to substrates of MAO-A, MAO-B, and SSAO with apparent inhibition constants (Ki) of 19.46, 5.38, and 1.84 microM.

Bacteria of the Burkholderia cepacia complex are cyanogenic under biofilm and colonial growth conditions.

BACKGROUND: The Burkholderia cepacia complex (Bcc) is a collection of nine genotypically distinct but phenotypically similar species. They show wide ecological diversity and include species that are used for promoting plant growth and bio-control as well species that are opportunistic pathogens of vulnerable patients. Over recent years the Bcc have emerged as problematic pathogens of the CF lung. Pseudomonas aeruginosa is another important CF pathogen. It is able to synthesise hydrogen cyanide (HCN), a potent inhibitor of cellular respiration. We have recently shown that HCN production by P. aeruginosa may have a role in CF pathogenesis. This paper describes an investigation of the ability of bacteria of the Bcc to make HCN. RESULTS: The genome of Burkholderia cenocepacia has 3 putative HCN synthase encoding (hcnABC) gene clusters. B. cenocepacia and all 9 species of the Bcc complex tested were able to make cyanide at comparable levels to P. aeruginosa, but only when grown surface attached as colonies or during biofilm growth on glass beads. In contrast to P. aeruginosa and other cyanogenic bacteria, cyanide was not detected during planktonic growth of Bcc strains. CONCLUSION: All species in the Bcc are cyanogenic when grown as surface attached colonies or as biofilms.

Mechanism of hcnA mRNA recognition in the Gac/Rsm signal transduction pathway of Pseudomonas fluorescens.

In the plant-beneficial bacterium Pseudomonas fluorescens CHA0, the expression of antifungal exoproducts is controlled by the GacS/GacA two-component system. Two RNA binding proteins (RsmA, RsmE) ensure effective translational repression of exoproduct mRNAs. At high cell population densities, GacA induces three small RNAs (RsmX, RsmY, RsmZ) which sequester both RsmA and RsmE, thereby relieving translational repression. Here we systematically analyse the features that allow the RNA binding proteins to interact strongly with the 5' untranslated leader mRNA of the P. fluorescens hcnA gene (encoding hydrogen cyanide synthase subunit A). We obtained evidence for three major RsmA/RsmE recognition elements in the hcnA leader, based on directed mutagenesis, RsmE footprints and toeprints, and in vivo expression data. Two recognition elements were found in two stem-loop structures whose existence in the 5' leader region was confirmed by lead(II) cleavage analysis. The third recognition element, which overlapped the hcnA Shine-Dalgarno sequence, was postulated to adopt either an open conformation, which would favour ribosome binding, or a stem-loop structure, which may form upon interaction with RsmA/RsmE and would inhibit access of ribosomes. Effective control of hcnA expression by the Gac/Rsm system appears to result from the combination of the three appropriately spaced recognition elements.

Cloning and characterization of Arabidopsis and Brassica juncea flavin-containing amine oxidases.

Polyamines (PAs) are low molecular weight metabolites involved in various physiological and developmental processes in eukaryotic and prokaryotic cells. The cellular PA level is regulated in part by the action of amine oxidases (AOs) including copper diamine oxidases (DAOs) and flavoprotein polyamine oxidases (PAOs). In this study, the isolation and characterization of flavin amine oxidases (FAOs) from Brassica juncea (BJFAO) and Arabidopsis (ATFAO1) are reported that were clustered in the same group as polyamine oxidases from maize (MPAO) and barley (BPAO1) and monoamine oxidases from mammalian species. ATFAO1 was temporally and spatially regulated in Arabidopsis and showed distinct expression patterns in response to different stress treatments. To investigate the in vivo function of FAO, transgenic Arabidopsis plants expressing sense, antisense, and double-stranded BJFAO RNAs were generated and those with altered activity of FAOs were selected for further characterization. It was found that the shoot regeneration response in transgenic plants was significantly affected by the modulated PA levels corresponding to FAO activities. Tissues that originated from transgenic plants with down-regulated FAO activity were highly regenerative, while those from transgenic plants with upregulated FAO activity were poorly regenerative. The shoot regeneration capacity in these transgenic plants was related to the levels of individual PAs, suggesting that FAO affects shoot regeneration by regulating cellular PAs. Furthermore, it was found that the effect of FAO activity on shoot regeneration was exerted downstream of the Enhancer of Shoot Regeneration (ESR1) gene, which may function in a branch of the cytokinin signalling pathway.

Genetic diversity and biocontrol potential of fluorescent pseudomonads producing phloroglucinols and hydrogen cyanide from Swiss soils naturally suppressive or conducive to Thielaviopsis basicola-mediated black root rot of tobacco.

Pseudomonas populations producing the biocontrol compounds 2,4-diacetylphloroglucinol (Phl) and hydrogen cyanide (HCN) were found in the rhizosphere of tobacco both in Swiss soils suppressive to Thielaviopsis basicola and in their conducive counterparts. In this study, a collection of Phl+ HCN+Pseudomonas isolates from two suppressive and two conducive soils were used to assess whether suppressiveness could be linked to soil-specific properties of individual pseudomonads. The isolates were compared based on restriction analysis of the biocontrol genes phlD and hcnBC, enterobacterial repetitive intergenic consensus (ERIC)-PCR profiling and their biocontrol ability. Restriction analyses of phlD and hcnBC yielded very concordant relationships between the strains, and suggested significant population differentiation occurring at the soil level, regardless of soil suppressiveness status. This was corroborated by high strain diversity (ERIC-PCR) within each of the four soils and among isolates harboring the same phlD or hcnBC alleles. No correlation was found between the origin of the isolates and their biocontrol activity in vitro and in planta. Significant differences in T. basicola inhibition were however evidenced between the isolates when they were grouped according to their biocontrol alleles. Moreover, two main Pseudomonas lineages differing by the capacity to produce pyoluteorin were evidenced in the collection. Thus, Phl+ HCN+ pseudomonads from suppressive soils were not markedly different from those from nearby conducive soils. Therefore, as far as biocontrol pseudomonads are concerned, this work yields the hypothesis that the suppressiveness of Swiss soils may rely on the differential effects of environmental factors on the expression of key biocontrol genes in pseudomonads rather than differences in population structure of biocontrol Pseudomonas subcommunities or the biocontrol potential of individual Phl+ HCN+ pseudomonad strains.