Identification of novel members of the bacterial azoreductase family in 'Pseudomonas aeruginosa'

Azoreductases are a family of diverse enzymes found in many pathogenic bacteria as well as distant homologues being present in eukarya. In addition to having azoreductase activity these enzymes are also suggested to have NAD(P)H quinone oxidoreductase activity which leads to a proposed role in plant pathogenesis. Azoreductases have also been suggested to play role in the mammalian pathogenesis of Pseudomonas aeruginosa. In view of the importance of P. aeruginosa as a pathogen, we therefore characterised recombinant enzymes following expression of a group of putative azoreductase genes from P. aeruginosa expressed in Escherichia coli . The enzymes include members of the "Arsenic resistance protein H" (ArsH), "tryptophan repressor binding protein A" (WrbA), "modulator of drug activity B" (MdaB) and YieF families. The ArsH, MdaB and YieF family members all show azoreductase and NAD(P)H quinone oxidoreductase activities. In contrast, WrbA is the first enzyme to show NAD(P)H quinone oxidoreductase activity but does not reduce any of the 11 azo compounds tested under a wide range of conditions. These studies will allow further investigation of the possible role of these enzymes in the pathogenesis of P. aeruginosa

Identification of NAD(P)H Quinone Oxidoreductase Activity in Azoreductases from P. aeruginosa: Azoreductases and NAD(P)H Quinone Oxidoreductases Belong to the Same FMN-Dependent Superfamily of Enzymes

Water soluble quinones are a group of cytotoxic anti-bacterial compounds that are secreted by many species of plants, invertebrates, fungi and bacteria. Studies in a number of species have shown the importance of quinones in response to pathogenic bacteria of the genus Pseudomonas. Two electron reduction is an important mechanism of quinone detoxification as it generates the less toxic quinol. In most organisms this reaction is carried out by a group of flavoenzymes known as NAD(P)H quinone oxidoreductases. Azoreductases have previously been separate from this group, however using azoreductases from Pseudomonas aeruginosa we show that they can rapidly reduce quinones. Azoreductases from the same organism are also shown to have distinct substrate specificity profiles allowing them to reduce a wide range of quinones. The azoreductase family is also shown to be more extensive than originally thought, due to the large sequence divergence amongst its members. As both NAD(P)H quinone oxidoreductases and azoreductases have related reaction mechanisms it is proposed that they form an enzyme superfamily. The ubiquitous and diverse nature of azoreductases alongside their broad substrate specificity, indicates they play a wide role in cellular survival under adverse conditions

Azoreductases : genes and proteins in ' Pseudomonas aeruginosa '

Pseudomonas aeruginosa is one of the primary causes of opportunistic infections in humans and it is associated with both acute and chronic infections in immunocompromised individuals. This bacterium is extremely resistant to many antibiotics, making the treatments against this pathogen often unsuccessful. Azoreductases, a family of enzymes involved in the reduction of azo compounds and quinones, are found in many bacterial species including P. aeruginosa. Although the enzymatic activity of three of these enzymes has been extensively characterized, their physiological role remains unclear. In this study, the enzymatic activity as well as the effect on physiological processes such as swarming motility, biofilm formation and antibiotic resistance of known and putative azoreductase proteins from P. aeruginosa PAO1 have been investigated. Five putative azoreductase genes from P. aeruginosa PAO1 (pa0949, pa1204, pa2280, pa2580 and pa4975) were cloned and four of these (pa0949, pa1204, pa2280 and pa2580) were over expressed in E. coli strains. Recombinant proteins were purified and biochemically characterized showing the presence of FAD bound to PA1204 and PA2580 proteins. Enzymatic reaction conditions were established for each protein by determining the preferred cofactor and reductant (flavin and NAD(P)H) used by each protein. Higher reduction rates were obtained using FAD for PA1204 and PA2580, and FMN for PA0949 and PA2280, whereas NADPH was always the preferred reductant for all of the enzymes tested. Substrate specificity studies performed with azo compounds and quinones showed that PA1204, PA2280 and PA2580 recombinant proteins can reduce both classes of substrate, with higher reduction rates with quinones, whereas the recombinant PA0949 protein showed to reduce only quinone substrates. Investigation of the role of azoreductase genes on P. aeruginosa PAO1 motility, biofilm formation and antibiotic resistance was conducted using single gene transposon mutants for each of the genes paazor1, paazor2, paazor3, pa0949, pa1204, pa2280, pa2580 and pa4975. Motility analysis demonstrated greater swarming for all mutants tested compared with wild type, although both wild type and mutants showed the same growth rate. Similarly, all mutants showed higher biofilm production compared with the wild type in a short term period (24 hours), whereas no differences were observed after a longer biofilm production period (48 hours). The MICs and MBCs of several antibiotics were determined, showing that, in presence of fluoroquinolones, P. aeruginosa PAO1 azoreductase mutants exhibit higher growth inhibition (up to 127-fold) and reduced survival (up to 7 fold) compared with wild type. This suggests that azoreductase genes (or gene products) may be involved in the P. aeruginosa PAO1 resistance to antibiotic treatments, and in particular to fluoroquinolones. The findings prove that the proteins PA0949, PA1204, PA2280 and PA2580 have similar features and enzymatic functions to the already characterized paAzoR1-3 from P. aeruginosa PAO1. Therefore, these can be included in the family of azo- and quinone- oxidoreductase enzymes. The data presented here on the antibiotic resistance strongly suggest a role for azoreductase gene products in antimicrobial resistance in P. aeruginosa. These original findings provide a springboard for further investigation of azoreductases as novel targets for antimicrobial agents for this pathogen

Processing and refinement statistics for paAzoR1 complexed with AQN and UQ1.

a<p>Numbers in parentheses are for the highest resolution shell.</p>b<p>Ramachandran statistics were calculated using MolProbity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098551#pone.0098551-Chen2" target="_blank">[37]</a>.</p

Quinone substrate specificity profiles of paAzoR1 (A), paAzoR2 (B) and paAzoR3 (C).

<p>All rates are normalised relative to the maximum rate of NADPH (paAzoR1) or NADH (paAzoR2/paAzoR3) oxidation observed for that enzyme. These maximum rates were as follows: paAzoR1 and paAzoR2 reducing Bzq 0.57 mM.s⁻¹.mg of enzyme⁻¹ and 7.78 mM.s⁻¹.mg of enzyme⁻¹ respectively and paAzoR3 reducing Plu 16.8 mM.s⁻¹.mg of enzyme⁻¹. All rates represent the average of three measurements ± standard deviation.</p

Binding of paAzoR1 to AQN and UQ1.

<p>Binding of paAzoR1 to AQN (A) and UQ1 (B). AQN is shown with pink carbon atoms, UQ1 with grey carbon atoms and FMN with yellow carbon atoms. The green mesh is unbiased positive difference density (<i>F_o</i>-<i>F_c</i> map) contoured at 3σ while the blue mesh is the refined 2<i>F_o</i>-<i>F_c</i> map at 1σ. A red dashed line signifies a hydrogen bond. Residues interacting with the ligands are labelled. (A) is based upon PDB 4N65, while (B) is based upon PDB 4N9Q.</p

Phylogenetic tree illustrating relationships between known and suspected azoreductases from <i>P. aeruginosa</i> PAO1.

<p>An unrooted bootstrap consensus maximum parsimony tree was generated using 500 replicates in Mega 6 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098551#pone.0098551-Tamura1" target="_blank">[78]</a>. This tree was based upon a sequence alignment of 33 proteins using Muscle <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098551#pone.0098551-Edgar1" target="_blank">[79]</a>. The tree was then rooted so that it is consistent with three previously published trees <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098551#pone.0098551-Patridge1" target="_blank">[74]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098551#pone.0098551-Vasiliou1" target="_blank">[80]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098551#pone.0098551-Chen3" target="_blank">[81]</a>. Enzymes in red are from <i>P. aeruginosa</i>, those in green are other bacterial enzymes, those in blue are mammalian enzymes, those in purple are plant enzymes, those in pink are fungal enzymes and those in yellow are archeal. PA0949, PA1204, PA1224, PA1225, PA2280, PA2580, and PA4975 are proteins from <i>P. aeruginosa</i>. ecAzoR, bsAzoR, efAzoR and rsAzoR are azoreductases from <i>E. coli</i>, <i>Bacillus subtilis</i>, <i>Enterococcus faecalus</i> and <i>Rhodobacter sphaeroides</i>. hNQO1 hNQO2, rNQO1 and rNQO2 are human and rat azoreductases. xaAzoR is a flavin independent azoreductase from <i>X. azovorans</i>. ecMdaB, ecYieF and ecWrbA are NAD(P)H quinone oxidoreductases from <i>E. coli</i>. afNQO, pnNQO, tmNQO, pcNQO and atNQO are NAD(P)H quinone oxidoreductases from <i>Archaeoglobus fulgidus</i>, <i>Paracoccus denitrificans</i>, <i>Triticum monococcum</i>, <i>Phanerochaete chrysosporium</i> and <i>Arabidopsis thaliana</i> respectively. ArsH is an azoreductase from <i>Sinorhizobium meliloti</i>. dgFlav and ecFlav are flavodoxins from <i>Desulfovibrio gigas</i> and <i>E. coli</i> respectively. shNQO, reNQO and erNQO are uncharacterised proteins from <i>Staphylococcus haemolyticus</i>, <i>Ralstonia eutropha</i> and <i>Erwinia chrysanthemi.</i></p

Structures of azoreductase homologues overlaid onto paAzoR1.

<p>(A) Structures of ArsH (PDB: 2Q62 - lilac), ecMdaB (PDB: 2B3D – coral) and hNQO2 (PDB: 2QR2 - brown) overlaid onto paAzoR1. (B) Structures of PA0949 (PDB: 1ZWL - red) and PA1204 (PDB: 1×77 - green) overlaid onto paAzoR1. In all cases, one monomer of the dimer is shown. In both cases, paAzoR1 is in blue (PDB: 2V9C). All alignments were carried out via secondary structure matching and images were generated in CCP4MG <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098551#pone.0098551-McNicholas1" target="_blank">[82]</a>.</p

Structural and functional information on all azoreductase homologues.

a<p>PDB codes for structures, ND not determined. Where particular enzymatic activities have been observed a reference is provided, otherwise no reference to the activity has been reported.</p

Distribution of azoreductase-like enzymes across <i>Pseudomonas</i> species.

<p>All values are given as percentage identity to the protein from <i>P. aeruginosa</i> PAO1. Homologues are defined as having >45% sequence identity based upon Blastp alignments. The strains whose genomes searched are as follows:</p>a<p>pf0–1,</p>b<p>KT2440,</p>c<p>ATCC 17588,</p>d<p>ATCC 13867,</p>e<p>30–84 and ^fDC3000.</p><p>The genes encoding paAzoR1, ^gpaAzoR2^h and paAzoR3ⁱ are marked.</p