The redox‐sensing regulator YodB senses quinones and diamide via a thiol‐disulfide switch in Bacillus subtilis

The MarR/DUF24‐type repressor YodB controls the azoreductase AzoR1, the nitroreductase YodC and the redox‐sensing regulator Spx in response to quinones and diamide in Bacillus subtilis. Previously, we showed using a yodBCys6‐Ala mutant that the conserved Cys6 apparently contributes to the DNA‐binding activity of YodB in vivo. Here, we present data that mutation of Cys6 to Ser led to a form of the protein that was reduced in redox‐sensing in response to diamide and 2‐methylhydroquinone (MHQ) in vivo. DNA‐binding experiments indicate that YodB is regulated by a reversible thiol‐modification in response to diamide and MHQ in vitro. Redox‐regulation of YodB involves Cys6‐Cys101' intermolecular disulfide formation by diamide and quinones in vitro. Diagonal Western blot analyses confirm the formation of intersubunit disulfides in YodB in vivo that require the conserved Cys6 and either of the C‐terminal Cys101' or Cys108' residues. This study reveals a thiol‐disulfide switch model of redox‐regulation for the YodB repressor to sense electrophilic compounds in vivo.     

Characterisation of the flavin-free oxygen-tolerant azoreductase from Xenophilus azovorans KF46F in comparison to flavin-containing azoreductases

The flavin-free azoreductase from Xenophilus azovorans KF46F (AzoB), which has been the very first characterized oxygen-tolerant azoreductase, was analyzed in comparison to various recently described flavin-containing azoreductases from different bacterial sources. Sequence comparisons demonstrated that the azoreductase from X. azovorans KF46F is a member of the NmrA family of proteins and demonstrates 30% sequence identity with a NADPH-dependent quinone oxidoreductase from Escherichia coli (encoded by ytfG). In contrast, it was found that the flavin-containing azoreductases from E. coli OY1-2 (AZR), Bacillus sp. OY1-2 (AZR) and related azoreductases all belong to the FMN_red superfamily of enzymes. The substrate specificity of AzoB was reanalyzed in respect to the recently characterized flavin-containing azoreductases, and it was found that purified AzoB converted in addition to different ortho-hydroxy azo compounds [such as Orange II = 1-(4′-sulfophenylazo)-2-naphthol] also the simple non-hydroxylated non-sulfonated azo dye Methyl Red (4′-dimethylaminoazobenzene-2-carboxylic acid), but no indications for the conversion of quinones were obtained. Significant differences were observed in the substrate specificities between AzoB and the flavin-containing azoreductases. The kinetic analysis of the turn-over of Orange II by AzoB suggested an ordered bireactant reaction mechanism which was different from the ping-pong mechanism suggested for the flavin-containing azoreductases.     

Three-dimensional structure of AzoR from Escherichia coli. An oxidereductase conserved in microorganisms

The crystal structure of AzoR (azoreductase) has been determined in complex with FMN for two different crystal forms at 1.8 and 2.2 A resolution. AzoR is an oxidoreductase isolated from Escherichia coli as a protein responsible for the degradation of azo compounds. This enzyme is an FMN-dependent NADH-azoreductase and catalyzes the reductive cleavage of azo groups by a ping-pong mechanism. The structure suggests that AzoR acts in a homodimeric state forming the two identical catalytic sites to which both monomers contribute. The structure revealed that each monomer of AzoR has a flavodoxin-like structure, without the explicit overall amino acid sequence homology. Superposition of the structures from the two different crystal forms revealed the conformational change and suggested a mechanism for accommodating substrates of different size. Furthermore, comparison of the active site structure with that of NQO1 complexed with substrates provides clues to the possible substrate-binding mechanism of AzoR.     

Identification, Isolation and characterization of a novel azoreductase from Clostridium perfringens

Azo dyes are used widely in the textile, pharmaceutical, cosmetic and food industries as colorants and are often sources of environmental pollution. There are many microorganisms that are able to reduce azo dyes by use of an azoreductase enzyme. It is through the reduction of the azo bonds of the dyes that carcinogenic metabolites are produced thereby a concern for human health. The field of research on azoreductases is growing, but there is very little information available on azoreductases from strict anaerobic bacteria. In this study, the azoreductase gene was identified in Clostridium perfringens, a pathogen that is commonly found in the human intestinal tract. C. perfringens shows high azoreductase activity, especially in the presence of the common dye Direct Blue 15. A gene that encodes for a flavoprotein was isolated and expressed in Escherichia coli, and further purified and tested for azoreductase activity. The azoreductase (known as AzoC) was characterized by enzymatic reaction assays using different dyes. AzoC activity was highest in the presence of two cofactors, NADH and FAD. A strong cofactor effect was shown with some dyes, as dye reduction occurred without the presence of the AzoC (cofactors alone). AzoC was shown to perform best at a pH of 9, at room temperature, and in an anaerobic environment. Enzyme kinetics studies suggested that the association between enzyme and substrate is strong. Our results show that AzoC from C. perfringens has azoreductase activity.     

Azoreductase activity of anaerobic bacteria isolated from human intestinal microflora.

A plate assay was developed for the detection of anaerobic bacteria that produce azoreductases. With this plate assay, 10 strains of anaerobic bacteria capable of reducing azo dyes were isolated from human feces and identified as Eubacterium hadrum (2 strains), Eubacterium spp. (2 species), Clostridium clostridiiforme, a Butyrivibrio sp., a Bacteroides sp., Clostridium paraputrificum, Clostridium nexile, and a Clostridium sp. The average rate of reduction of Direct Blue 15 dye (a dimethoxybenzidine-based dye) in these strains ranged from 16 to 135 nmol of dye per min per mg of protein. The enzymes were inactivated by oxygen. In seven isolates, a flavin compound (riboflavin, flavin adenine dinucleotide, or flavin mononucleotide) was required for azoreductase activity. In the other three isolates and in Clostridium perfringens, no added flavin was required for activity. Nondenaturing polyacrylamide gel electrophoresis showed that each bacterium expressed only one azoreductase isozyme. At least three types of azoreductase enzyme were produced by the different isolates. All of the azoreductases were produced constitutively and released extracellularly.     

Azoreductase activity of anaerobic bacteria isolated from human intestinal microflora

A plate assay was developed for the detection of anaerobic bacteria that produce azoreductases. With this plate assay, 10 strains of anaerobic bacteria capable of reducing azo dyes were isolated from human feces and identified as Eubacterium hadrum (2 strains), Eubacterium spp. (2 species), Clostridium clostridiiforme, a Butyrivibrio sp., a Bacteroides sp., Clostridium paraputrificum, Clostridium nexile, and a Clostridium sp. The average rate of reduction of Direct Blue 15 dye (a dimethoxybenzidine-based dye) in these strains ranged from 16 to 135 nmol of dye per min per mg of protein. The enzymes were inactivated by oxygen. In seven isolates, a flavin compound (riboflavin, flavin adenine dinucleotide, or flavin mononucleotide) was required for azoreductase activity. In the other three isolates and in Clostridium perfringens, no added flavin was required for activity. Nondenaturing polyacrylamide gel electrophoresis showed that each bacterium expressed only one azoreductase isozyme. At least three types of azoreductase enzyme were produced by the different isolates. All of the azoreductases were produced constitutively and released extracellularly.     

Nitric oxide promotes strong cytotoxicity of phenolic compounds against Escherichia coli: the influence of antioxidant defenses

The induction of mutagenic and cytotoxic effects by simple phenolics, including catechol (CAT), 3,4-dihydroxyphenylacetic acid (DOPAC), hydroquinone (HQ), and 2,5-dihydroxyphenylacetic (homogentisic) acid (HGA), appears to occur through an oxidative mechanism based on the ability of these compounds to undergo autoxidation, leading to quinone formation with the production of reactive oxygen species. This is supported by the detection of such adverse effects in plate assays using Escherichia coli tester strains deficient in the OxyR function, but not in OxyR(+) strains. The OxyR protein is a redox-sensitive regulator of genes encoding antioxidant enzymes including catalase and alkyl hydroperoxide reductase, which would eliminate hydrogen peroxide. Methyl-substituted phenolics such as 4-methylcatechol (MCAT) and methylhydroquinone (MHQ) produced, in addition to oxidative toxicity, marked cytotoxic effects against OxyR(+) cells, thus revealing a mechanism of toxicity not mediated by hydrogen peroxide that could involve quinones and quinone methides arising from MCAT and MHQ oxidation. Quinone compounds could also be responsible for the enhanced cytotoxicity of certain phenolics when combined with a nitric oxide (NO(*)) donor such as diethylamine/NO (DEA/NO). Phenolics scavenge NO(*) and, in turn, NO(*) oxidizes phenolics to form their quinone derivatives. In OxyR(+) cells, where the oxidative toxicity is inhibited, DEA/NO promoted exceptional increases in the cytotoxicity of CAT and 3,4-dihydroxycinnamic (caffeic) acid (CAF), which both exhibited very low oxidative cytotoxicity, as well as in that of MCAT, HQ, and MHQ. In contrast, DEA/NO failed to promote toxicity by DOPAC and HGA, probably due to their ability to undergo oxidative polymerization, leading to the formation of melanins. Spectroscopic studies demonstrated quinone generation from the oxidation of CAF, HQ, and MHQ by DEA/NO. The o-quinone derived from CAF was rather unstable and decomposed during its isolation. For the generation of toxic quinones, e.g., to be used as therapeutic agents producing antitumor or antibacterial effects, the isolation step could be avoided with the method proposed. It combines quinone precursors, i.e. phenolic compounds, with an oxidant such as NO(*).     

Purification and partial characterization of two azoreductases from Shigella dysenteriae Type 1

Two azoreductases (I and II) were purified to homogeneity from extracts of Shigella dysenteriae (type 1). Azoreductase I was a dimer of identical subunits of M(r) 28,000, whereas azoreductase II was a monomer of 11,000 M(r). Both were flavoproteins, each containing 1 mol of FMN per mol enzyme. Both NADH and NADPH functioned as electron donors for the azoreductases. Azoreductase I used Ponceau SX, Tartrazine, Amaranth and Orange II as substrates. Azoreductase II utilized all the dyes except Amaranth.     

Determining the molecular mechanism of inactivation by chemical modification of triosephosphate isomerase from the human parasite Giardia lamblia: a study for antiparasitic drug design

Giardiasis, the most prevalent intestinal parasitosis in humans, is caused by Giardia lamblia. Current drug therapies have adverse effects on the host, and resistant strains against these drugs have been reported, demonstrating an urgent need to design more specific antigiardiasic drugs. ATP production in G. lamblia depends mainly on glycolysis; therefore, all enzymes of this pathway have been proposed as potential drug targets. We previously demonstrated that the glycolytic enzyme triosephosphate isomerase from G. lamblia (GlTIM), could be completely inactivated by low micromolar concentrations of thiol-reactive compounds, whereas, in the same conditions, the activity of human TIM (HuTIM) was almost unaltered. We found that the chemical modification (derivatization) of at least one Cys, of the five Cys residues per monomer in GlTIM, causes this inactivation. In this study, structural and functional studies were performed to describe the molecular mechanism of GlTIM inactivation by thiol-reactive compounds. We found that the Cys222 derivatization is responsible for GlTIM inactivation; this information is relevant because HuTIM has a Cys residue in an equivalent position (Cys217). GlTIM inactivation is associated with a decrease in ligand affinity, which affects the entropic component of ligand binding. In summary, this work describes a mechanism of inactivation that has not been previously reported for TIMs from other parasites and furthermore, we show that the difference in reactivity between the Cys222 in GlTIM and the Cys217 in HuTIM, indicates that the surrounding environment of each Cys residue has unique structural differences that can be exploited to design specific antigiardiasic drugs.