Genome-Scale Investigation of the Regulation of azoR Expression in Escherichia coli Using Computational Analysis and Transposon Mutagenesis.

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.

In Silico Comparison of Bioactive Compounds Characterized from Azadirachta indica with an FDA-Approved Drug against Schistosomal Agents: New Insight into Schistosomiasis Treatment.

The burden of human schistosomiasis, a known but neglected tropical disease in Sub-Saharan Africa, has been worrisome in recent years. It is becoming increasingly difficult to tackle schistosomiasis with praziquantel, a drug known to be effective against all Schistosoma species, due to reports of reduced efficacy and resistance. Therefore, this study seeks to investigate the antischistosomal potential of phytochemicals from Azadirachta indica against proteins that have been implicated as druggable targets for the treatment of schistosomiasis using computational techniques. In this study, sixty-three (63) previously isolated and characterized phytochemicals from A. indica were identified from the literature and retrieved from the PubChem database. In silico screening was conducted to assess the inhibitory potential of these phytochemicals against three receptors (Schistosoma mansoni Thioredoxin glutathione reductase, dihydroorotate dehydrogenase, and Arginase) that may serve as therapeutic targets for schistosomiasis treatment. Molecular docking, ADMET prediction, ligand interaction, MMGBSA, and molecular dynamics simulation of the hit compounds were conducted using the Schrodinger molecular drug discovery suite. The results show that Andrographolide possesses a satisfactory pharmacokinetic profile, does not violate the Lipinski rule of five, binds with favourable affinity with the receptors, and interacts with key amino acids at the active site. Importantly, its interaction with dihydroorotate dehydrogenase, an enzyme responsible for the catalysis of the de novo pyrimidine nucleotide biosynthetic pathway rate-limiting step, shows a glide score and MMGBSA of -10.19 and -45.75 Kcal/mol, respectively. In addition, the MD simulation shows its stability at the active site of the receptor. Overall, this study revealed that Andrographolide from Azadirachta indica could serve as a potential lead compound for the development of an anti-schistosomal drug.

A Redox-Regulated, Heterodimeric NADH:cinnamate Reductase in Vibrio ruber.

Genes of putative reductases of α,ß-unsaturated carboxylic acids are abundant among anaerobic and facultatively anaerobic microorganisms, yet substrate specificity has been experimentally verified for few encoded proteins. Here, we co-produced in Escherichia coli a heterodimeric protein of the facultatively anaerobic marine bacterium Vibrio ruber (GenBank SJN56019 and SJN56021; annotated as NADPH azoreductase and urocanate reductase, respectively) with Vibrio cholerae flavin transferase. The isolated protein (named Crd) consists of the sjn56021-encoded subunit CrdB (NADH:flavin, FAD binding 2, and FMN bind domains) and an additional subunit CrdA (SJN56019, a single NADH:flavin domain) that interact via their NADH:flavin domains (Alphafold2 prediction). Each domain contains a flavin group (three FMNs and one FAD in total), one of the FMN groups being linked covalently by the flavin transferase. Crd readily reduces cinnamate, p-coumarate, caffeate, and ferulate under anaerobic conditions with NADH or methyl viologen as the electron donor, is moderately active against acrylate and practically inactive against urocanate and fumarate. Cinnamates induced Crd synthesis in V. ruber cells grown aerobically or anaerobically. The Crd-catalyzed reduction started by NADH demonstrated a time lag of several minutes, suggesting a redox regulation of the enzyme activity. The oxidized enzyme is inactive, which apparently prevents production of reactive oxygen species under aerobic conditions. Our findings identify Crd as a regulated NADH-dependent cinnamate reductase, apparently protecting V. ruber from (hydroxy)cinnamate poisoning.

Design of a cofactor self-sufficient whole-cell biocatalyst for enzymatic asymmetric reduction via engineered metabolic pathways and multi-enzyme cascade.

NAD(P)H-dependent oxidoreductases are crucial biocatalysts for synthesizing chiral compounds. Yet, the industrial implementation of enzymatic redox reactions is often hampered by an insufficient supply of expensive nicotinamide cofactors. Here, a cofactor self-sufficient whole-cell biocatalyst was developed for the enzymatic asymmetric reduction of 2-oxo-4-[(hydroxy)(-methyl)phosphinyl] butyric acid (PPO) to L-phosphinothricin (L-PPT). The endogenous NADP+ pool was significantly enhanced by regulating Preiss-Handler pathway toward NAD(H) synthesis and, in the meantime, introducing NAD kinase to phosphorylate NAD(H) toward NADP+. The intracellular NADP(H) concentration displayed a 2.97-fold increase with the strategy compared with the wild-type strain. Furthermore, a recombinant multi-enzyme cascade biocatalytic system was constructed based on the Escherichia coli chassis. In order to balance multi-enzyme co-expression levels, the strategy of modulating rate-limiting enzyme PmGluDH by RBS strengths regulation successfully increased the catalytic efficiency of PPO conversion. Finally, the cofactor self-sufficient whole-cell biocatalyst effectively converted 300 mM PPO to L-PPT in 2 h without the need to add exogenous cofactors, resulting in a 2.3-fold increase in PPO conversion (%) from 43% to 100%, with a high space-time yield of 706.2 g L-1 d-1 and 99.9% ee. Overall, this work demonstrates a technological example for constructing a cofactor self-sufficient system for NADPH-dependent redox biocatalysis.

GRIM19 deficiency aggravates metabolic disorder and ovarian dysfunction in PCOS.

CONTEXT: Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women. Retinoid-interferon-induced mortality 19 (GRIM19) is a functional component of mitochondrial complex I that plays a role in cellular energy metabolism. However, the role of GRIM19 in the pathogenesis of PCOS is still unclear. OBJECTIVE: To investigate the role of GRIM19 in the pathogenesis of PCOS. DESIGN: We first measured the expression of GRIM19 in human granulosa cells (hGCs) from patients with and without PCOS (n = 16 per group), and then established a PCOS mouse model with WT and Grim19+/- mice for in vivo experiments. Glucose uptake-related genes RAC1 and GLUT4 and energy metabolism levels in KGN cells were examined in vitro by knocking down GRIM19 in the cell lines. Additionally, ovulation-related genes such as p-ERK1/2, HAS2, and PTX3 were also studied to determine their expression levels. RESULTS: GRIM19 expression was reduced in hGCs of PCOS patients, which was negatively correlated with BMI and serum testosterone level. Grim19+/- mice with PCOS exhibited a markedly anovulatory phenotype and disturbed glycolipid metabolism. In vitro experiments, GRIM19 deficiency inhibited the RAC1/GLUT4 pathway, reducing insulin-stimulated glucose uptake in KGN cells. Moreover, GRIM19 deficiency induced mitochondrial dysfunction, defective glucose metabolism, and apoptosis. In addition, GRIM19 deficiency suppressed the expression of ovulation-related genes in KGN cells, which was regulated by dihydrotestosterone mediated androgen receptor. CONCLUSIONS: GRIM19 deficiency may mediate ovulation and glucose metabolism disorders in PCOS patients. Our results suggest that GRIM19 may be a new target for diagnosis and treatment.

Cloning and characterization of FMN-dependent azoreductases from textile industry effluent identified through metagenomic sequencing.

Azo dyes, when released untreated in the environment, cause detrimental effects on flora and fauna. Azoreductases are enzymes capable of cleaving commercially used azo dyes, sometimes in less toxic by-products which can be further degraded via synergistic microbial cometabolism. In this study, azoreductases encoded by FMN1 and FMN2 genes were screened from metagenome shotgun sequences generated from the samples of textile dye industries' effluents, cloned, expressed, and evaluated for their azo dye decolorization efficacy. At pH 7 and 45°C temperature, both recombinant enzymes FMN1 and FMN2 were able to decolorize methyl red at 20 and 100 ppm concentrations, respectively. FMN2 was found to be more efficient in decolorization/degradation of methyl red than FMN1. This study offers valuable insights into the possible application of azoreductases to reduce the environmental damage caused by azo dyes, with the hope of contributing to sustainable and eco-friendly practices for the environment management. This enzymatic approach offers a promising solution for the bioremediation of textile industrial effluents. However, the study acknowledges the need for further process optimization to enhance the efficacy of these enzymes in large-scale applications.Implications: The study underscores the environmental hazards associated with untreated release of azo dyes into the environment and emphasizes the potential of azoreductases, specifically those encoded by FMN1 and FMN2 genes, to mitigate the detrimental effects. The study emphasizes the ongoing commitment to refining and advancing the enzymatic approach for the bioremediation of azo dye-containing effluents, marking a positive stride toward more sustainable industrial practices.

GRIM-19 deficiency promotes macrophage polarization to the M1 phenotype partly through glycolysis in unexplained recurrent spontaneous abortion†.

The occurrence of unexplained recurrent spontaneous abortion (URSA) is closely related to immune system disorders, however, the underlying mechanisms remain unclear. The purpose of this study was to investigate the expression of GRIM-19 in URSA and the possible pathogenesis of URSA according to macrophage polarization. Here, we showed that GRIM-19 was downregulated in the uterine decidual macrophages of patients with URSA and that GRIM-19 downregulation was accompanied by increased M1 macrophage polarization. Furthermore, the expression levels of glycolytic enzymes were substantially enhanced in the uterine decidual macrophages of URSA patients, and glycolysis in THP-1-derived macrophages was further enhanced by the downregulation of GRIM-19. Additionally, the increase of M1 macrophages resulting from the loss of GRIM-19 was significantly reversed in cells treated with 2-deoxy-D-glucose (2-DG, an inhibitor of glycolysis). To provide more direct evidence, GRIM-19 deficiency was shown to promote macrophage polarization to the M1 phenotype in GRIM-19+/- mouse uteri. Overall, our study provides evidence that GRIM-19 deficiency may play a role in regulating macrophage polarization in URSA, and that glycolysis may participate in this process.

Visualization of an Endogenous Mitochondrial Azoreductase Activity under Normoxic Conditions Using a Naphthalimide Azo-Based Fluorogenic Probe.

In this paper, we demonstrate the existence of an endogenous mitochondrial azoreductase (AzoR) activity that can induce the cleavage of N═N double bonds of azobenzene compounds under normoxic conditions. To this end, 100% OFF-ON azo-based fluorogenic probes derived from 4-amino-1,8-naphthalimide fluorophores were synthesized and evaluated. The in vitro study conducted with other endogenous reducing agents of the cell, including reductases, demonstrated both the efficacy and the selectivity of the probe for AzoR. Confocal experiments with the probe revealed an AzoR activity in the mitochondria of living cells under normal oxygenation conditions, and we were able to demonstrate that this endogenous AzoR activity appears to be expressed at different levels across different cell lines. This discovery provides crucial information for our understanding of the biochemical processes occurring within the mitochondria. It thus contributes to a better understanding of its function, which is implicated in numerous pathologies.

Fragment Merging, Growing, and Linking Identify New Trypanothione Reductase Inhibitors for Leishmaniasis.

Trypanothione reductase (TR) is a suitable target for drug discovery approaches against leishmaniasis, although the identification of potent inhibitors is still challenging. Herein, we harnessed a fragment-based drug discovery (FBDD) strategy to develop new TR inhibitors. Previous crystallographic screening identified fragments 1-3, which provided ideal starting points for a medicinal chemistry campaign. In silico investigations revealed critical hotspots in the TR binding site, guiding our structure- and ligand-based structure-actvity relationship (SAR) exploration that yielded fragment-derived compounds 4-14. A trend of improvement in Leishmania infantum TR inhibition was detected along the optimization and confirmed by the crystal structures of 9, 10, and 14 in complex with Trypanosoma brucei TR. Compound 10 showed the best TR inhibitory profile (Ki = 0.2 µM), whereas 9 was the best one in terms of in vitro and ex vivo activity. Although further fine-tuning is needed to improve selectivity, we demonstrated the potentiality of FBDD on a classic but difficult target for leishmaniasis.

Revealing the degrading-possibility of methyl red by two azoreductases of Anoxybacillus sp. PDR2 based on molecular docking.

Azo dyes, as the most widely used synthetic dyes, are considered to be one of the culprits of water resources and environmental pollution. Anoxybacillus sp. PDR2 is a thermophilic bacterium with the ability to degrade azo dyes, whose genome contains two genes encoding azoreductases (named AzoPDR2-1 and AzoPDR2-2). In this study, through response surface methodology (RSM), when the initial pH, inoculation volume and Mg2+ addition amount were 7.18, 10.72% and 0.1 g/L respectively, the decolorization rate of methyl red (MR) (200 mg/L) could reach its maximum (98.8%). The metabolites after biodegradation were detected by UV-Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), and liquid chromatography mass spectrometry (LC-MS/MS), indicating that MR was successfully decomposed into 4-aminobenzoic acid and other small substrates. In homologous modeling, it was found that both azoreductases were flavin-dependent azoreductases, and belonged to the α/ß structure, using the Rossmann fold. In their docking results with the cofactor flavin mononucleotide (FMN), FMN bound to the surface of the protein dimer. Nicotinamide adenine dinucleotide (NADH) was superimposed on the plane of the pyrazine ring between FMN and the activity pocket of protein. Besides, both azoreductase complexes (azoreductase-FMN-NADH) exhibited a substrate preference for MR. Asn104 and Tyr74 played an important role in the combination of the azoreductase AzoPDR2-1 complex and the azoreductase AzoPDR2-2 complex with MR, respectively. This provided assistance for studying the mechanism of azoreductase biodegradation of azo dyes in thermophilic bacteria.

Coupling of NAD(P)H:FMN-oxidoreductase and luciferase from luminous bacteria in a viscous medium: Finding the weakest link in the chain.

The study aims at revealing the mechanisms of the viscous medium effects on the kinetic features of NAD(P)H:FMN-oxidoreductase from luminous bacteria (Red), which are exhibited in a single enzyme assay and in coupling with bacterial luciferase (BLuc). Different concentrations of glycerol and sucrose were used to vary the medium viscosity. The activity of Red, alone and in the presence of BLuc, was analyzed, as well as BLuc activity in the presence of Red, whereas in the absence of BLuc, the Red activity was suppressed in viscous medium, and in the presence of BLuc, the increase in Red activity was observed at low glycerol concentrations (5-20 wt%). The interaction of glycerol and sucrose with Red substrates FMN and NADH was studied using absorption spectroscopy and molecular dynamics. Glycerol was found to form hydrogen bonds with the phosphate groups of the substrates, unlike sucrose. A mechanism for the activation of Red in the presence of BLuc in glycerol solutions through the acceleration of FMN reoxidation was proposed. Thus, it was concluded that, under the conditions used, the weakest link of the coupled enzyme system BLuc-Red in viscous medium is the FMN concentration, which depends on Red activity and the medium viscosity.

Protein N-myristoylation plays a critical role in the mitochondrial localization of human mitochondrial complex I accessory subunit NDUFB7.

The present study examined human N-myristoylated proteins that specifically localize to mitochondria among the 1,705 human genes listed in MitoProteome, a mitochondrial protein database. We herein employed a strategy utilizing cellular metabolic labeling with a bioorthogonal myristic acid analog in transfected COS-1 cells established in our previous studies. Four proteins, DMAC1, HCCS, NDUFB7, and PLGRKT, were identified as N-myristoylated proteins that specifically localize to mitochondria. Among these proteins, DMAC1 and NDUFB7 play critical roles in the assembly of complex I of the mitochondrial respiratory chain. DMAC1 functions as an assembly factor, and NDUFB7 is an accessory subunit of complex I. An analysis of the intracellular localization of non-myristoylatable G2A mutants revealed that protein N-myristoylation occurring on NDUFB7 was important for the mitochondrial localization of this protein. Furthermore, an analysis of the role of the CHCH domain in NDUFB7 using Cys to Ser mutants revealed that it was essential for the mitochondrial localization of NDUFB7. Therefore, the present results showed that NDUFB7, a vital component of human mitochondrial complex I, was N-myristoylated, and protein N-myrisotylation and the CHCH domain were both indispensable for the specific targeting and localization of NDUFB7 to mitochondria.

In Silico Exploration of the Trypanothione Reductase (TryR) of L. mexicana.

Human leishmaniasis is a neglected tropical disease which affects nearly 1.5 million people every year, with Mexico being an important endemic region. One of the major defense mechanisms of these parasites is based in the polyamine metabolic pathway, as it provides the necessary compounds for its survival. Among the enzymes in this route, trypanothione reductase (TryR), an oxidoreductase enzyme, is crucial for the Leishmania genus' survival against oxidative stress. Thus, it poses as an attractive drug target, yet due to the size and features of its catalytic pocket, modeling techniques such as molecular docking focusing on that region is not convenient. Herein, we present a computational study using several structure-based approaches to assess the druggability of TryR from L. mexicana, the predominant Leishmania species in Mexico, beyond its catalytic site. Using this consensus methodology, three relevant pockets were found, of which the one we call σ-site promises to be the most favorable one. These findings may help the design of new drugs of trypanothione-related diseases.

Identification of novel coenzyme Q10 biosynthetic proteins Coq11 and Coq12 in Schizosaccharomyces pombe.

Coenzyme Q (CoQ) is an essential component of the electron transport system in aerobic organisms. CoQ10 has ten isoprene units in its quinone structure and is especially valuable as a food supplement. However, the CoQ biosynthetic pathway has not been fully elucidated, including synthesis of the p-hydroxybenzoic acid (PHB) precursor to form a quinone backbone. To identify the novel components of CoQ10 synthesis, we investigated CoQ10 production in 400 Schizosaccharomyces pombe gene-deleted strains in which individual mitochondrial proteins were lost. We found that deletion of coq11 (an S. cerevisiae COQ11 homolog) and a novel gene designated coq12 lowered CoQ levels to ∼4% of that of the WT strain. Addition of PHB or p-hydroxybenzaldehyde restored the CoQ content and growth and lowered hydrogen sulfide production of the Δcoq12 strain, but these compounds did not affect the Δcoq11 strain. The primary structure of Coq12 has a flavin reductase motif coupled with an NAD+ reductase domain. We determined that purified Coq12 protein from S. pombe displayed NAD+ reductase activity when incubated with ethanol-extracted substrate of S. pombe. Because purified Coq12 from Escherichia coli did not exhibit reductase activity under the same conditions, an extra protein is thought to be necessary for its activity. Analysis of Coq12-interacting proteins by LC-MS/MS revealed interactions with other Coq proteins, suggesting formation of a complex. Thus, our analysis indicates that Coq12 is required for PHB synthesis, and it has diverged among species.

Amino acids can deplete ATP and impair nitric oxide detoxification by Escherichia coli.

Nitric oxide (·NO) is a prevalent antimicrobial that is known to damage iron-containing enzymes in amino acid (AA) biosynthesis pathways. With Escherichia coli, ·NO is detoxified in aerobic environments by Hmp, which is an enzyme that is synthesized de novo in response to ·NO. With this knowledgebase, it is expected that the availability of AAs in the extracellular environment would enhance ·NO detoxification, because AAs would foster translation of Hmp. However, we observed that ·NO detoxification by E. coli was far slower in populations grown and treated in the presence of AAs (AA+) in comparison to those grown and stressed in the absence of AAs (AA-). Further experiments revealed that AA+ populations had difficulty translating proteins under ·NO stress, and that ·NO activated the stringent response in AA+ populations. Additional work revealed significant ATP depletion in ·NO-stressed AA+ cultures that far exceeded that of ·NO-stressed AA- populations. Transcription, translation, and RelA were not found to be significant contributors to the ATP depletion observed, whereas AA import was implicated as a significant ATP consumption pathway. Alleviating ATP depletion while maintaining access to AAs partially restored ·NO detoxification, which suggested that ATP depletion contributed to the translational difficulties observed in ·NO-stressed AA+ populations. These data reveal an unexpected interaction within the ·NO response network of E. coli that stimulates a stringent response by RelA in conditions where AAs are plentiful.

A predicted structure of NADPH Oxidase 1 identifies key components of ROS generation and strategies for inhibition.

NADPH oxidase 1 (NOX1) is primarily expressed in epithelial cells and responsible for local generation of reactive oxygen species (ROS). By specifically manipulating the local redox microenvironment, NOX1 actively engages in epithelial immunity, especially in colorectal and pulmonary epithelia. To unravel the structural basis of NOX1 engaged epithelial immune processes, a predicted structure model was established using RaptorX deep learning models. The predicted structure model illustrates a 6-transmembrane domain structure, a FAD binding domain, and an NADPH binding/NOXO1 interacting region. The substrate/cofactor binding scheme with respect to this proposed model highly correlates with published reports and is verified in our site-directed mutagenesis assays. An electron transport chain, from NADPH to FAD and the two heme groups, was well supported by the predicted model. Through molecular docking analysis of various small molecule NOX1 inhibitors and subsequent experimental validation, we identified pronounced active sites for potent NOX1 inhibition. Specifically, LEU60, VAL71, MET181, LEU185, HIS208, PHE211, TYR214, and TYR280 in the transmembrane domain form an active pocket for insertion of the small molecule inhibitors to inhibit electron transfer between the heme groups, thus affecting extracellular ROS generation. Altogether, our study provides structural information to help elucidate the role of NOX1 in epithelial generation of ROS and sheds light on the development of therapeutics for NOX1 related illnesses.

NADH dehydrogenases are the predominant phenazine reductases in the electron transport chain of Pseudomonas aeruginosa.

Phenazines are redox-active secondary metabolites produced by diverse bacteria including the opportunistic pathogen Pseudomonas aeruginosa. Extracellular electron transfer via phenazines enhances anaerobic survival by serving as an electron sink for glucose catabolism. However, the specific phenazine reductase(s) used to support this catabolism are unknown. Because electron transport chain components have been previously implicated in phenazine reduction, we sought to determine which of them possess phenazine reductase activity. We show that phenazine-1-carboxamide (PCN) and pyocyanin (PYO) are reduced at the highest rate by cells and are localized to the cell envelope while reduced. Using a coupled genetic and biochemical approach, we show that phenazine reductase activity in membrane fractions is attributable to the three NADH dehydrogenases present in P. aeruginosa and that their order of phenazine reductase activity is Nqr > Nuo > Ndh. In mutants possessing only one functional NADH dehydrogenase, whole cell reduction rates of PCN, but not PYO, recapitulate the pattern of biochemical results, implying that PYO reduction is predominantly occurring in the cytosol. Lastly, we show that ubiquinone rapidly and non-enzymatically oxidizes reduced phenazines, demonstrating that phenazines have the capability to serve in a redox loop between the NADH and ubiquinone pools, a finding that carries bioenergetic implications.

Nonreducing terminal chimeric isomaltomegalosaccharide and its integration with azoreductase for the remediation of soil-contaminated lipophilic azo dyes.

Lipophilic azo dyes are practically water-insoluble, and their dissolution by organic solvents and surfactants is harmful to biological treatment with living cells and enzymes. This study aimed to evaluate the feasibility of a newly synthesized nonreducing terminal chimeric isomaltomegalosaccharide (N-IMS) as a nontoxic solubilizer of four simulated lipophilic azo dye wastes for enzymatic degradation. N-IMS bearing a helical α-(1 â†’ 4)-glucosidic segment derived from a donor substrate α-cyclodextrin was produced by a coupling reaction of cyclodextrin glucanotransferase. Inclusion complexing by N-IMS overcame the solubility issue with equilibrium constants of 1786-242 M-1 (methyl yellow > ethyl red > methyl red > azo violet). Circular dichroism spectra revealed the axial alignment of the aromatic rings in the N-IMS cavity, while UV-visible absorption quenching revealed that the azo bond of methyl yellow was particularly induced. Desorption of the dyes from acidic and neutral soils was specific to aqueous organic over alkali extraction. The dissolution kinetics of the incorporated dyes followed a sigmoid pattern facilitating the subsequent decolorization process with azoreductase. It was demonstrated that after soil extraction, the solid dyes dissolved with N-IMS assistance and spontaneously digested by coupled azoreductase/glucose dehydrogenase (for a cofactor regeneration system) with the liberation of the corresponding aromatic amine.

Gut Microbiome-Wide Search for Bacterial Azoreductases Reveals Potentially Uncharacterized Azoreductases Encoded in the Human Gut Microbiome.

The human gut is home to trillions of microorganisms that are responsible for the modification of many orally administered drugs, leading to a wide range of therapeutic outcomes. Prodrugs bearing an azo bond are designed to treat inflammatory bowel disease and colorectal cancer via microbial azo reduction, allowing for topical application of therapeutic moieties to the diseased tissue in the intestines. Despite the inextricable link between microbial azo reduction and the efficacy of azo prodrugs, the prevalence, abundance, and distribution of azoreductases have not been systematically examined across the gut microbiome. Here, we curated and clustered amino acid sequences of experimentally confirmed bacterial azoreductases and conducted a hidden Markov model-driven homolog search for these enzymes across 4644 genome sequences present in the representative Unified Human Gastrointestinal Genomes collection. We identified 1958 putative azo-reducing species, corroborating previous findings that azo reduction appears to be a ubiquitous function of the gut microbiome. However, through a systematic comparison of predicted and confirmed azo-reducing strains, we hypothesize the presence of uncharacterized azoreductases in 25 prominent strains of the human gut microbiome. Finally, we confirmed the azo reduction of Acid Orange 7 by multiple strains of Fusobacterium nucleatum, Bacteroides fragilis, and Clostridium clostridioforme Together, these results suggest the presence and activity of many uncharacterized azoreductases in the human gut microbiome and motivate future studies aimed at characterizing azoreductase genes in prominent members of the human gut microbiome. SIGNIFICANCE STATEMENT: This work systematically examined the prevalence, abundance, and distribution of azoreductases across the healthy and inflammatory bowel disease human gut microbiome, revealing potentially uncharacterized azoreductase genes. It also confirmed the reduction of Acid Orange 7 by strains of Fusobacterium nucleatum, Bacteroides fragilis, and Clostridium clostridioforme.

A natural fusion of flavodiiron, rubredoxin, and rubredoxin oxidoreductase domains is a self-sufficient water-forming oxidase of Trichomonas vaginalis.

Microaerophilic pathogens such as Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis have robust oxygen consumption systems to detoxify oxygen and maintain intracellular redox balance. This oxygen consumption results from H2O-forming NADH oxidase (NOX) activity of two distinct flavin-containing systems: H2O-forming NOXes and multicomponent flavodiiron proteins (FDPs). Neither system is membrane bound, and both recycle NADH into oxidized NAD+ while simultaneously removing O2 from the local environment. However, little is known about the specific contributions of these systems in T. vaginalis. In this study, we use bioinformatics and biochemical analyses to show that T. vaginalis lacks a NOX-like enzyme and instead harbors three paralogous genes (FDPF1-3), each encoding a natural fusion product between the N-terminal FDP, central rubredoxin (Rb), and C-terminal NADH:Rb oxidoreductase domains. Unlike a "stand-alone" FDP that lacks Rb and oxidoreductase domains, this natural fusion protein with fully populated flavin redox centers directly accepts reducing equivalents of NADH to catalyze the four-electron reduction of oxygen to water within a single polypeptide with an extremely high turnover. Furthermore, using single-particle cryo-EM, we present structural insights into the spatial organization of the FDP core within this multidomain fusion protein. Together, these results contribute to our understanding of systems that allow protozoan parasites to maintain optimal redox balance and survive transient exposure to oxic conditions.