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ImportanceThis work has broad relevance due to the ubiquity of dyes containing azo bonds in food and drugs. We report that azo dyes can be degraded by human gut bacteria through both enzymatic and nonenzymatic mechanisms, even from a single gut bacterial species. Furthermore, we revealed that environmental factors, oxygen, and L-Cysteine control the ability of E. coli to degrade azo dyes due to their impacts on bacterial transcription and metabolism. These results open up new opportunities to manipulate the azoreductase activity of the gut microbiome through the manipulation of host diet, suggest that azoreductase potential may be altered in patients suffering from gastrointestinal disease, and highlight the importance of studying bacterial enzymes for drug metabolism in their natural cellular and ecological context. more...

Bacterial azoreductases are enzymes that catalyze the reduction of ingested or industrial azo dyes. Although azoreductase genes have been well identified and characterized, the regulation of their expression has not been systematically investigated. To determine how different factors affect the expression of azoR, we extracted and analyzed transcriptional data from the Gene Expression Omnibus (GEO) resource, then confirmed computational predictions by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Results showed that azoR expression was lower with higher glucose concentration, agitation speed, and incubation temperature, but higher at higher culture densities. Co-expression and clustering analysis indicated ten genes with similar expression patterns to azoR: melA, tpx, yhbW, yciK, fdnG, fpr, nfsA, nfsB, rutF, and chrR (yieF). In parallel, constructing a random transposon library in E. coli K-12 and screening 4320 of its colonies for altered methyl red (MR)-decolorizing activity identified another set of seven genes potentially involved in azoR regulation. Among these genes, arsC, relA, plsY, and trmM were confirmed as potential azoR regulators based on the phenotypic decolorization activity of their transposon mutants, and the expression of arsC and relA was confirmed, by qRT-PCR, to significantly increase in E. coli K-12 in response to different MR concentrations. Finally, the significant decrease in azoR transcription upon transposon insertion in arsC and relA (as compared to its expression in wild-type E. coli) suggests their probable involvement in azoR regulation. In conclusion, combining in silico analysis and random transposon mutagenesis suggested a set of potential regulators of azoR in E. coli. [ABSTRACT FROM AUTHOR] more...

Abstract The present study aimed to investigate the decolorization of various commercial dyes by azoreductases (AzrBmH21, AzrBmH22/3, and AzrBmH24/5) through bioinformatics means, comprising molecular docking, molecular dynamics simulation, and molecular mechanics Poisson–Boltzmann surface area (MM-PBSA). Therefore, four commercial dyes, namely acid orange 7, cresol red, methylene blue, and malachite green, were selected as potential targets for degradation by the above said azoreductases derived from Bacillus megaterium H2. The prediction of ligand binding or catalytic sites for AzrBmH21, AzrBmH22/3, and AzrBmH24/5 were performed using a machine learning algorithm based on the Prank Web and DeepSite chemoinformatic tool. This analysis revealed that several amino acids of AzrBmH2 interacted with the tested dyes, indicating the presence of distinct ligand-binding sites for AzrBmH2-dye complexes. Likewise, the binding affinity for AzrBmH21, AzrBmH22/3, and AzrBmH24/5 ranged from − 9.4 to − 5.5 kcal/mol, − 9.2 to − 5.4 kcal/mol, and − 9.0 to − 5.4 kcal/mol, respectively, with each complex stabilized at a minimum of 0–5 hydrogen bonds. MD simulations revealed stable AzrBmH2-dye complexes with RMSD and RMSF values ranging from 0.15 to 0.42 nm and 0.05 to 0.48 nm, respectively, with Rg values between 1.75 and 1.88 nm. MM-PBSA calculations indicated that the AzrBmH2–dye complexes, except for AzrBmH2–malachite green, exhibited the lowest binding energy (− 191.05 ± 7.08 to 314.19 ± 6.88 kcal/mol), with prevalent hydrophobic interactions (− 268.25 ± 12.25 to − 418.92 ± 29.45 kcal/mol) through van der Waals forces. Therefore, this study was able to highlight the potential role of enzymes, specifically azoreductases from Bacillus megaterium H2, in predicting the decolorization of commercial dyes. These findings could contribute to our understanding of the azoreductases’ mechanisms in bioremediation and for biotechnological applications. more...

The present study aimed to investigate the decolorization of various commercial dyes by azoreductases (AzrBmH21, AzrBmH22/3, and AzrBmH24/5) through bioinformatics means, comprising molecular docking, molecular dynamics simulation, and molecular mechanics Poisson–Boltzmann surface area (MM-PBSA). Therefore, four commercial dyes, namely acid orange 7, cresol red, methylene blue, and malachite green, were selected as potential targets for degradation by the above said azoreductases derived from Bacillus megaterium H2. The prediction of ligand binding or catalytic sites for AzrBmH21, AzrBmH22/3, and AzrBmH24/5 were performed using a machine learning algorithm based on the Prank Web and DeepSite chemoinformatic tool. This analysis revealed that several amino acids of AzrBmH2 interacted with the tested dyes, indicating the presence of distinct ligand-binding sites for AzrBmH2-dye complexes. Likewise, the binding affinity for AzrBmH21, AzrBmH22/3, and AzrBmH24/5 ranged from − 9.4 to − 5.5 kcal/mol, − 9.2 to − 5.4 kcal/mol, and − 9.0 to − 5.4 kcal/mol, respectively, with each complex stabilized at a minimum of 0–5 hydrogen bonds. MD simulations revealed stable AzrBmH2-dye complexes with RMSD and RMSF values ranging from 0.15 to 0.42 nm and 0.05 to 0.48 nm, respectively, with Rg values between 1.75 and 1.88 nm. MM-PBSA calculations indicated that the AzrBmH2–dye complexes, except for AzrBmH2–malachite green, exhibited the lowest binding energy (− 191.05 ± 7.08 to 314.19 ± 6.88 kcal/mol), with prevalent hydrophobic interactions (− 268.25 ± 12.25 to − 418.92 ± 29.45 kcal/mol) through van der Waals forces. Therefore, this study was able to highlight the potential role of enzymes, specifically azoreductases from Bacillus megaterium H2, in predicting the decolorization of commercial dyes. These findings could contribute to our understanding of the azoreductases' mechanisms in bioremediation and for biotechnological applications. [ABSTRACT FROM AUTHOR] more...

Food and drug products contain diverse and abundant small-molecule additives (excipients) with unclear impacts on human physiology, drug safety, and response. Here, we evaluate their potential impact on intestinal drug absorption. By screening 136 unique compounds for inhibition of the key intestinal transporter OATP2B1 we identified and validated 24 potent OATP2B1 inhibitors, characterized by higher molecular weight and hydrophobicity compared to poor or noninhibitors. OATP2B1 inhibitors were also enriched for dyes, including 8 azo (R-N=N-R') dyes. Pharmacokinetic studies in mice confirmed that FD&C Red No. 40, a common azo dye excipient and a potent inhibitor of OATP2B1, decreased the plasma level of the OATP2B1 substrate fexofenadine, suggesting that FD&C Red No. 40 has the potential to block drug absorption through OATP2B1 inhibition in vivo. However, the gut microbiomes of multiple unrelated healthy individuals as well as diverse human gut bacterial isolates were capable of inactivating the identified azo dye excipients, producing metabolites that no longer inhibit OATP2B1 transport. These results support a beneficial role for the microbiome in limiting the unintended effects of food and drug additives in the intestine and provide a framework for the data-driven selection of excipients. Furthermore, the ubiquity and genetic diversity of gut bacterial azoreductases coupled to experiments in conventionally raised and gnotobiotic mice suggest that variations in gut microbial community structure may be less important to consider relative to the high concentrations of azo dyes in food products, which have the potential to saturate gut bacterial enzymatic activity. more...

ABSTRACT The microbiome is an underappreciated contributor to intestinal drug metabolism with broad implications for drug efficacy and toxicity. While considerable progress has been made toward identifying the gut bacterial genes and enzymes involved, the role of environmental factors in shaping their activity remains poorly understood. Here, we focus on the gut bacterial reduction of azo bonds (R-N = N-R’), found in diverse chemicals in both food and drugs. Surprisingly, the canonical azoR gene in Escherichia coli was dispensable for azo bond reduction. Instead, azoreductase activity was controlled by the fumarate and nitrate reduction (fnr) regulator, consistent with a requirement for the anoxic conditions found within the gastrointestinal tract. Paired transcriptomic and proteomic analysis of the fnr regulon revealed that in addition to altering the expression of multiple reductases, FNR is necessary for the metabolism of L-Cysteine to hydrogen sulfide, enabling the degradation of azo bonds. Furthermore, we found that FNR indirectly regulates this process through the small noncoding regulatory RNA fnrS. Taken together, these results show how gut bacteria sense and respond to their intestinal environment to enable the metabolism of chemical groups found in both dietary and pharmaceutical compounds. IMPORTANCE This work has broad relevance due to the ubiquity of dyes containing azo bonds in food and drugs. We report that azo dyes can be degraded by human gut bacteria through both enzymatic and nonenzymatic mechanisms, even from a single gut bacterial species. Furthermore, we revealed that environmental factors, oxygen, and L-Cysteine control the ability of E. coli to degrade azo dyes due to their impacts on bacterial transcription and metabolism. These results open up new opportunities to manipulate the azoreductase activity of the gut microbiome through the manipulation of host diet, suggest that azoreductase potential may be altered in patients suffering from gastrointestinal disease, and highlight the importance of studying bacterial enzymes for drug metabolism in their natural cellular and ecological context. more...

It is necessary to develop appropriate approaches to eliminate para -nitrophenol (PNP) in our environment, because the pollutant is highly toxic and also able to persist in the environment. Previously, Pseudomonas sp. strain WBC-3 isolated from polluted soil was found to be able to use PNP as the sole carbon and nitrogen source, but not very efficiently. In this study, WBC-3 was shown to belong to Pseudomonas putida through de novo genome sequencing. To enhance its efficiency of PNP utilization, a mutant strain (PM1-33) with a significantly increased PNP degradation rate was obtained. Although no increase in the expression levels of known PNP catabolizing genes/proteins were observed between WBC-3 and PM1-33, the expression level of protein 1619 significantly increased in PM1-33. Deleting GM1619 in WBC-3 and PM1-33 caused decreased PNP degradation rates in both strains and eliminated the difference in PNP degradation between the two strains. Functional prediction using AlphaFold2 showed that protein1619 might bind to p -benzoquinone (BQ). Consequently, protein 1619 was biochemically characterized, confirming its ability to convert BQ into hydroquinone (HQ). Thus, a new protein involved in PNP degradation was identified, thereby adding new knowledge to bacterial PNP degradation pathways. • Pseudomonas sp. WBC-3 belongs to Pseudomonas putida through de novo sequencing. • A mutant strain (PM1-33) with accelerated PNP degradation was obtained. • Protein 1619 is essential for the accelerated PNP degradation rate in PM1-33. • Protein 1619 participates in PNP degradation by converting BQ into HQ. [ABSTRACT FROM AUTHOR] more...

Food and drug products contain diverse and abundant smallmolecule additives (excipients) with unclear impacts on human physiology, drug safety, and response. Here, we evaluate their potential impact on intestinal drug absorption. By screening 136 unique compounds for inhibition of the key intestinal transporter OATP2B1 we identified and validated 24 potent OATP2B1 inhibitors, characterized by higher molecular weight and hydrophobicity compared to poor or noninhibitors. OATP2B1 inhibitors were also enriched for dyes, including 8 azo (R−N=N−R′) dyes. Pharmacokinetic studies in mice confirmed that FD&C Red No. 40, a common azo dye excipient and a potent inhibitor of OATP2B1, decreased the plasma level of the OATP2B1 substrate fexofenadine, suggesting that FD&C Red No. 40 has the potential to block drug absorption through OATP2B1 inhibition in vivo. However, the gut microbiomes of multiple unrelated healthy individuals as well as diverse human gut bacterial isolates were capable of inactivating the identified azo dye excipients, producing metabolites that no longer inhibit OATP2B1 transport. These results support a beneficial role for the microbiome in limiting the unintended effects of food and drug additives in the intestine and provide a framework for the data-driven selection of excipients. Furthermore, the ubiquity and genetic diversity of gut bacterial azoreductases coupled to experiments in conventionally raised and gnotobiotic mice suggest that variations in gut microbial community structure may be less important to consider relative to the high concentrations of azo dyes in food products, which have the potential to saturate gut bacterial enzymatic activity. [ABSTRACT FROM AUTHOR] more...

Abstract Background Nitroreductases, NAD(P)H dependent flavoenzymes, are found in most of bacterial species. Even if Enterococcus faecalis strains seems to present such activity because of their sensitivity to nitrofurans, no enzyme has been described. Nitroreductases were separated of others reductases due to their capacity to reduce nitro compounds. They are further classified based on their preference in cofactor: NADH and/or NADPH. However, recently, azoreductases have been studied for their strong activity on nitro compounds, especially nitro pro-drugs. This result suggests a crossing in azo and nitro reductase activities. For the moment, no nitroreductase was demonstrated to possess azoreductase activity. But due to sequence divergence and activity specificity linked to substrates, activity prediction is not evident and biochemical characterisation remains necessary. Identifying enzymes active on these two classes of compounds: azo and nitro is of interest to consider a common physiological role. Results Four putative nitroreductases, EF0404, EF0648, EF0655 and EF1181 from Enterococcus faecalis V583 were overexpressed as his-tagged recombinant proteins in Escherichia coli and purified following a native or a denaturing/renaturing protocol. EF0648, EF0655 and EF1181 showed nitroreductase activity and their cofactor preferences were in agreement with their protein sequence phylogeny. EF0404 showed both nitroreductase and azoreductase activity. Interestingly, the biochemical characteristics (substrate and cofactor specificity) of EF0404 resembled the properties of the known azoreductase AzoA. But its sequence matched within nitroreductase group, the same as EF0648. Conclusions We here demonstrate nitroreductase activity of the putative reductases identified in the Enterococcus faecalis V583 genome. We identified the first nitroreductase able to reduce directly an azo compound, while its protein sequence is close to others nitroreductases. Consequently, it highlights the difficulty in classifying these enzymes solely on the basis of protein sequence alignment and hereby the necessity to experimentally demonstrate the activity. The results provide additional data to consider a broader functionality of these reductases. more...

Background: Nitroreductases, NAD(P)H dependent flavoenzymes, are found in most of bacterial species. Even if Enterococcus faecalis strains seems to present such activity because of their sensitivity to nitrofurans, no enzyme has been described. Nitroreductases were separated of others reductases due to their capacity to reduce nitro compounds. They are further classified based on their preference in cofactor: NADH and/or NADPH. However, recently, azoreductases have been studied for their strong activity on nitro compounds, especially nitro pro-drugs. This result suggests a crossing in azo and nitro reductase activities. For the moment, no nitroreductase was demonstrated to possess azoreductase activity. But due to sequence divergence and activity specificity linked to substrates, activity prediction is not evident and biochemical characterisation remains necessary. Identifying enzymes active on these two classes of compounds: azo and nitro is of interest to consider a common physiological role. Results: Four putative nitroreductases, EF0404, EF0648, EF0655 and EF1181 from Enterococcus faecalis V583 were overexpressed as his-tagged recombinant proteins in Escherichia coli and purified following a native or a denaturing/renaturing protocol. EF0648, EF0655 and EF1181 showed nitroreductase activity and their cofactor preferences were in agreement with their protein sequence phylogeny. EF0404 showed both nitroreductase and azoreductase activity. Interestingly, the biochemical characteristics (substrate and cofactor specificity) of EF0404 resembled the properties of the known azoreductase AzoA. But its sequence matched within nitroreductase group, the same as EF0648. Conclusions: We here demonstrate nitroreductase activity of the putative reductases identified in the Enterococcus faecalis V583 genome. We identified the first nitroreductase able to reduce directly an azo compound, while its protein sequence is close to others nitroreductases. Consequently, it highlights the difficulty in classifying these enzymes solely on the basis of protein sequence alignment and hereby the necessity to experimentally demonstrate the activity. The results provide additional data to consider a broader functionality of these reductases. [ABSTRACT FROM AUTHOR] more...

Azoreductase 1 from Pseudomonas aeruginosa strain PAO1 (paAzoR1) catalyses the activation of the prodrug balsalazide and reduces the azo dye methyl red using reduced nicotinamide adenine dinucleotide cofactor as an electron donor. To investigate the mechanism of the enzyme, a Y131F mutation was introduced and the enzymic properties of the mutant were compared with those of the wild-type enzyme. The crystallographic structure of the mutant with methyl red bound was solved at 2.1 Å resolution and compared with the wild-type structure. Tyr131 is important in the architecture of the active site but is not essential for enzymic activity. [ABSTRACT FROM AUTHOR] more...

Azoreductases require NAD(P)H to reduce azo dyes but the high cost of NAD(P)H limits its application. Formate dehydrogenase (FDH) allows NAD(P) + recycling and therefore, the fusion of these two biocatalysts seems promising. This study investigated the changes to the fusion protein involving azoreductase (AzoRo) of Rhodococcus opacus 1CP and FDH (FDH C23S and FDH C23SD195QY196H ) of Candida boidinii in different positions with His-tag as the linker. The position affected enzyme activities as AzoRo activity decreased by 20-fold when it is in the N-terminus of the fusion protein. FDH C23S +AzoRo was the most active construct and was further characterized. Enzymatic activities of FDH C23S +AzoRo decreased compared to parental enzymes but showed improved substrate scope - accepting bulkier dyes. Moreover, pH has an influence on the stability and activity of the fusion protein because at pH 6 (pH that is suboptimal for FDH), the dye reduction decreased to more than 50 % and this could be attributed to the impaired NADH supply for the AzoRo part., (© 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.) more...

PpAzoR, an FMN-dependent NADPH azoreductase from Pseudomonas putida MET94, has been crystallized using the sitting-drop vapour-diffusion technique. The crystals diffracted to 1.6 Å resolution using synchrotron radiation and belonged to the orthorhombic space group F222, with unit-cell parameters a = 72.1, b = 95.5, c = 146.1 Å. Data sets were collected from the native protein to 2.2 Å resolution using in-house equipment and to 1.6 Å resolution using synchrotron radiation and the three-dimensional structure was determined by the molecular-replacement method. [ABSTRACT FROM AUTHOR] more...

Azoreductases from Bacillus sp. B29 are NADH-dependent flavoenzymes which contain a flavin mononucleotide (FMN) as a prosthetic group and exist as homodimers composed of 23 kDa subunits. These enzymes catalyze the reductive degradation of various azo compounds by a ping-pong mechanism. In order to determine the structure-function relationship of the azo-dye reduction mechanism, an X-ray crystallographic study of azoreductases was performed. Selenomethionine-labelled AzrA (SeMet-AzrA) and AzrC were crystallized by the hanging-drop vapour-diffusion method. A crystal of SeMet-AzrA diffracted to 2.0 Å resolution and was determined to belong to space group P212121, with unit-cell parameters a = 56.9, b = 69.0, c = 105.4 Å. The native crystals of AzrC belonged to space group C2, with unit-cell parameters a = 192.0, b = 56.6, c = 105.5 Å, β = 115.7°, and diffracted to 2.21 Å resolution. [ABSTRACT FROM AUTHOR] more...

Gut microorganisms metabolize azobenzene compounds (Ph 1 -N=N-Ph 2 ) into free aniline products (Ph 1 -NH 2 +H 2 N-Ph 2 ), a process that has been largely investigated to reduce dyes residues in the textile industry. However, the action of bacterial core enzymes such as azoreductases (AzoR) might also help to deliver prodrugs that become active when they reach the colonic region, a mechanism with potential applications for the treatment of inflammatory bowel disease (IBD) and colorectal cancer. So far, three azo-bonded prodrugs of 5-aminosalicylic acid (5-ASA), for example, sulfasalazine, olsalazine and balsalazide, have been used for colon-targeted delivery. The present contribution describes the first rational design of a novel azobenzene prodrug thanks to a computational approach, with a focus on linking 5-ASA to another approved anti-inflammatory drug. The resulting prodrugs were assessed for their degradation upon AzoR action. Replacing the original carriers by irsogladine is found to improve action., (© 2021 Wiley-VCH GmbH.) more...

Azo dyes are major synthetic dyestuffs with one or more azo bonds and are widely used for various industrial purposes. The biodegradation of residual azo dyes via azoreductase-catalyzed cleavage is very efficient as the initial step of wastewater treatment. The structures of the complexes of azoreductases with various substrates are therefore indispensable to understand their substrate specificity and catalytic mechanism. In this study, the crystal structures of AzrA and of AzrC complexed with Cibacron Blue (CB) and the azo dyes Acid Red 88 (AR88) and Orange I (OI) were determined. As an inhibitor/analogue of NAD(P)H, CB was located on top of flavin mononucleotide (FMN), suggesting a similar binding manner as NAD(P)H for direct hydride transfer to FMN. The structures of the AzrC-AR88 and AzrC-OI complexes showed two manners of binding for substrates possessing a hydroxy group at the ortho or the para position of the azo bond, respectively, while AR88 and OI were estimated to have a similar binding affinity to AzrC from ITC experiments. Although the two substrates were bound in different orientations, the hydroxy groups were located in similar positions, resulting in an arrangement of electrophilic C atoms binding with a proton/electron-donor distance of ∼3.5 Å to N5 of FMN. Catalytic mechanisms for different substrates are proposed based on the crystal structures and on site-directed mutagenesis analysis. more...