Application of a Pulsed Electric Field to Modify the Free Energy of the Active Site of an Azoreductase

The Gibbs free energy is strongly related to the stability and catalytic function of an enzyme through the energetic changes that occur in the chemical reactions the enzyme catalyzes. In this in silico study, a pulsed electric field was applied to an azoreductase, and its effect on the Gibbs free energy of molecular docking with two dyes was measured. We propose that certain stimuli from a pulsed electric field favor the structural stability of the enzyme by promoting an arrangement in the active site, potentially leading to an enhancement of enzymatic activity overall.

Biodegradation of azo dyes by genetically engineered azoreductase

A azoreductase gene with 537 bp was obtained by PCR amplification from Rhodobacter sphaeroides AS1 1737 The enzyme, with a molecular weight of 18 7 kD, was efficiently expressed in Escherichia coli and its biodegradation characteristics for azo dyes were investigated. Furthermore, the reaction kinetics and mechanism of azo dyes catalyzed by the genetically engineered azoreductase were studied in detail. The presence of a hydrazo-intermediate was identified, which provided a convincing evidence for the assumption that azo dyes were degraded via an incomplete reduction stage.

Reaction mechanism of azoreductases suggests convergent evolution with quinone oxidoreductases

Azoreductases are involved in the bioremediation by bacteria of azo dyes found in waste water.In the gut flora,they activate azo pro-drugs,which are used for treatment of inflammatory bowel disease,releasing the active component 5-aminosalycilic acid.The bacterium P.aeruginosa has three azoreductase genes,paAzoR1,paAzoR2 and paAzoR3,which as recombinant enzymes have been shown to have different substrate specificities.The mechanism of azoreduction relies upon tautomerisation of the substrate to the hydrazone form.We report here the characterization of the P.aeruginosa azoreductase enzymes,including determining their thermostability,cofactor preference and kinetic constants against a range of their favoured substrates.The expression levels of these enzymes during growth of P.aeruginosa are altered by the presence of azo substrates.It is shown that enzymes that were originally described as azoreductases,are likely to act as NADH quinone oxidoreductases.The low sequence identities observed among NAD(P)H quinone oxidoreductase and azoreductase enzymes suggests convergent evolution.

Identification of a new azoreductase driven prodrug from bardoxolone methyl and 5-aminosalicylate for the treatment of colitis in mice

For local treatment of ulcerative colitis,a new azoreductase driven prodrug CDDO-AZO from bardoxolone methyl(CDDO-Me)and 5-aminosalicylate(5-ASA)was designed,synthesized and biologically evaluated.It is proposed that orally administrated CDDO-AZO is stable before reaching the colon,while it can also be triggered by the presence of azoreductase in the colon to fragment into CDDO-Me and 5-ASA,generating potent anti-colitis effects.Superior to olsalazine(OLS,a clinically used drug for ulcerative colitis)and CDDO-Me plus 5-ASA,CDDO-AZO significantly attenuated inflammatory colitis symptoms in DSS-induced chronic colitis mice,which suggested that CDDO-AZO may be a promising anti-ulcerative colitis agent.

Application of 2D fluorescence correlation method to investigate the dilution-induced heterogeneous distribution of the bound FMN in azoreductase

AzoR is a homodimeric,flavin mononucleotide（FMN）-containing,NADH-dependent azoreductase from Escherichia coli.In this paper,we investigated the effect of the concentration of both AzoR and R59 G on the spectral behavior of the bound FMN using two-dimensional fluorescence correlation spectra.Two cross peaks（530,490） and（580,530） were observed from the dilution-induced 2D asynchronous correlation map of wt AzoR,while only one cross peak appeared at（600,530） for R59 C mutant.This result indicated that the mutation at site 59 influenced the formation of dilution-induced intermediates.The specific activity of both AzoR and R59 G mutant was unaffected by dilution when the enzyme concentration is below 1 μmol/L,which suggested that no significant dissociation of FMN occurred at low concentrations.Additionally,in order to explore the origin of these intermediates,we carried out a2 D correlation analysis using excitation wavelength-dependent fluorescence emission spectroscopy.The results showed that there coexisted two types of FMN that emitted fluorescence at 530 nm and 500 nm,respectively.Taken together,these results suggested that the 2D method is a very powerful method to identify the heterogeneous distribution of the bound FMN in solution.

A near-infrared multifunctional fluorescent probe for hypoxia monitoring and tumor-targeted therapy

Hypoxia is one of the key characteristics of solid tumors. The over-expression of azoreductase resulting from hypoxia can be used as a target to visualize hypoxic levels and a trigger of the drug release system in tumor treatment. In this work, we developed a near-infrared fluorescent probe YLOD, composed of a near-infrared fluorophore, an azo bond, and an analogue of the anti-tumor drug melphalan. In the presence of azoreductase, YLOD displayed a red emission at 620 nm and released the anti-tumor drug concomitantly, thus achieving the integrated effects of hypoxic imaging and tumor treatment. Furthermore, YLOD successfully inhibited the growth of solid tumors during the tumor suppression experiments in nude mice. Considering all the results, YLOD emerges as a new fluorescence tool that can quickly determine the location and the edges of a tumor, showing concrete potential in clinical cancer treatment.

Decolorization of azo dyes by a salt-tolerant Staphylococcus cohnii strain isolated from textile wastewater

The salt-tolerant Staphylococcus cohnii strain, isolated from textile wastewater, has been found effective on decolorizing several kinds of azo dyes with different structures. The optimal conditions for azo dye acid red B （ARB） decolorization by S. cohnii were determined to be pH= 7.0 and 30℃. The decolorization efficiency increased with the increase of the salinity concentration, and around 90% of ARB （100mg.L-1） could be decolorized in 24 h when the salinity concentration was up to 50 g-L 1. Moreover, the strain could still decolorize 19% of ARB in 24 h even when the NaC1 concentration was increased to 150 g. L1. Meanwhile, the dependence of the specific decolorization rate by S. cohnii on the ARB concentration could be described with Michaelis-Menten kinetics （Kin = 585.7mg·L-1, Vmax = t09.8mg-g cell ·h ^-1）. The addition of quinone redox mediator, named 2-hydroxy-l,4-naphthoquinone and anthraqui- none-2,6-disulfonate, significantly accelerated the deco- lorization performance ofS. cohnii. Furtherly, the activities of azoreductase （0.55 μmol.mg protein^-1.min-1） and Nicotineamide adenine dinucleotide-dichlorophenol indophenol （NADH-DCIP） reductase （8.9μmol.mg proteinl.min-1） have been observed in the crude cell extracts of S. cohnii. The decolorization products of ARB were analyzed by HPLC-MS, and the results indicated the reductive pathway was responsible for azo dye decoloriza- tion by S. cohnii.