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Transgenic expression of bacterial nitroreductase (NTR) enzymes sensitizes eukaryotic cells to prodrugs such as metronidazole (MTZ), enabling selective cell-ablation paradigms that have expanded studies of cell function and regeneration in vertebrates. However, first-generation NTRs required confoundingly toxic prodrug treatments to achieve effective cell ablation, and some cell types have proven resistant. Here we used rational engineering and cross-species screening to develop an NTR variant, NTR 2.0, which exhibits ~100-fold improvement in MTZ-mediated cell-specific ablation efficacy, eliminating the need for near-toxic prodrug treatment regimens. NTR 2.0 therefore enables sustained cell-loss paradigms and ablation of previously resistant cell types. These properties permit enhanced interrogations of cell function, extended challenges to the regenerative capacities of discrete stem cell niches, and novel modeling of chronic degenerative diseases. Accordingly, we have created a series of bipartite transgenic reporter/effector resources to facilitate dissemination of NTR 2.0 to the research community. An engineered bacterial nitroreductase, NTR 2.0, improves chemically induced cell ablation, facilitating novel sustained ablation paradigms for testing the effects of chronic inflammation on regeneration, and modeling degenerative disease.

Most nitroaromatic compounds are toxic and mutagenic for living organisms, but some microorganisms have developed oxidative or reductive pathways to degrade or transform these compounds. Reductive pathways are based either on the reduction of the aromatic ring by hydride additions or on the reduction of the nitro groups to hydroxylamino and/or amino derivatives. Bacterial nitroreductases are flavoenzymes that catalyze the NAD(P)H-dependent reduction of the nitro groups on nitroaromatic and nitroheterocyclic compounds. Nitroreductases have raised a great interest due to their potential applications in bioremediation, biocatalysis, and biomedicine, especially in prodrug activation for chemotherapeutic cancer treatments. Different bacterial nitroreductases have been purified and their biochemical and kinetic parameters have been determined. The crystal structure of some nitroreductases have also been solved. However, the physiological role(s) of these enzymes remains unclear. Nitroreductase genes are widely spread within bacterial genomes, but are also found in archaea and some eukaryotic species. Although studies on regulation of nitroreductase gene expression are scarce, it seems that nitroreductase genes may be controlled by the MarRA and SoxRS regulatory systems that are involved in responses to several antibiotics and environmental chemical hazards and to specific oxidative stress conditions. This review covers the microbial distribution, types, biochemical properties, structure and regulation of the bacterial nitroreductases. The possible physiological functions and the biotechnological applications of these enzymes are also discussed.

E. coli nitroreductase A (NfsA) is a candidate for gene-directed prodrug cancer therapy using bioreductively activated nitroaromatic compounds (ArNO2). In this work, we determined the standard redox potential of FMN of NfsA to be −215 ± 5 mV at pH 7.0. FMN semiquinone was not formed during 5-deazaflavin-sensitized NfsA photoreduction. This determines the two-electron character of the reduction of ArNO2 and quinones (Q). In parallel, we characterized the oxidant specificity of NfsA with an emphasis on its structure. Except for negative outliers nitracrine and SN-36506, the reactivity of ArNO2 increases with their electron affinity (single-electron reduction potential, E17) and is unaffected by their lipophilicity and Van der Waals volume up to 386 Å. The reactivity of quinoidal oxidants is not clearly dependent on E17, but 2-hydroxy-1,4-naphthoquinones were identified as positive outliers and a number of compounds with diverse structures as negative outliers. 2-Hydroxy-1,4-naphthoquinones are characterized by the most positive reaction activation entropy and the negative outlier tetramethyl-1,4-benzoquinone by the most negative. Computer modelling data showed that the formation of H bonds with Arg15, Arg133, and Ser40, plays a major role in the binding of oxidants to reduced NfsA, while the role of the π–π interaction of their aromatic structures is less significant. Typically, the calculated hydride-transfer distances during ArNO2 reduction are smallwer than for Q. This explains the lower reactivity of quinones. Another factor that slows down the reduction is the presence of positively charged aliphatic substituents.

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.

Our inability to predict which mutations could result in antibiotic resistance has made it difficult to rapidly identify the emergence of resistance, identify pre-existing resistant populations, and manage our use of antibiotics to effectively treat patients and prevent or slow the spread of resistance. Here we investigated the potential for resistance against the new antitubercular nitroimidazole prodrugs pretomanid and delamanid to emerge in Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). Deazaflavin-dependent nitroreductase (Ddn) is the only identified enzyme within M. tuberculosis that activates these prodrugs, via an F420H2-dependent reaction. We show that the native menaquinone-reductase activity of Ddn is essential for emergence from hypoxia, which suggests that for resistance to spread and pose a threat to human health, the native activity of Ddn must be at least partially retained. We tested 75 unique mutations, including all known sequence polymorphisms identified among ~15,000 sequenced M. tuberculosis genomes. Several mutations abolished pretomanid and delamanid activation in vitro, without causing complete loss of the native activity. We confirmed that a transmissible M. tuberculosis isolate from the hypervirulent Beijing family already possesses one such mutation and is resistant to pretomanid, before being exposed to the drug. Notably, delamanid was still effective against this strain, which is consistent with structural analysis that indicates delamanid and pretomanid bind to Ddn differently. We suggest that the mutations identified in this work be monitored for informed use of delamanid and pretomanid treatment and to slow the emergence of resistance.

Diphenyl ether herbicides, typical globally used herbicides, threaten the agricultural environment and the sensitive crops. The microbial degradation pathways of diphenyl ether herbicides are well studied, but the nitroreduction of diphenyl ether herbicides by purified enzymes is still unclear. Here, the gene dnrA, encoding a nitroreductase DnrA responsible for the reduction of nitro to amino groups, was identified from the strain Bacillus sp. Za. DnrA had a broad substrate spectrum, and the Km values of DnrA for different diphenyl ether herbicides were 20.67 μM (fomesafen), 23.64 μM (bifenox), 26.19 μM (fluoroglycofen), 28.24 μM (acifluorfen), and 36.32 μM (lactofen). DnrA also mitigated the growth inhibition effect on cucumber and sorghum through nitroreduction. Molecular docking revealed the mechanisms of the compounds fomesafen, bifenox, fluoroglycofen, lactofen, and acifluorfen with DnrA. Fomesafen showed higher affinities and lower binding energy values for DnrA, and residue Arg244 affected the affinity between diphenyl ether herbicides and DnrA. This research provides new genetic resources and insights into the microbial remediation of diphenyl ether herbicide-contaminated environments.Key points• Nitroreductase DnrA transforms the nitro group of diphenyl ether herbicides.• Nitroreductase DnrA reduces the toxicity of diphenyl ether herbicides.• The distance between Arg244 and the herbicides is related to catalytic efficiency.

Nitroreductase (NTR), a class of flavin‐dependent redox enzymes, is a key biomarker for hypoxic tumors. Numerous fluorescent NTR probes have been developed to study hypoxia and associated tumors; however, they are reaction‐based and provide only static information on the accumulated enzyme activity at a given time. Reversible binding probes are needed to monitor the enzyme level in real time. Here, the first reversible binding probe is presented that selectively detects the active, reduced form of NTR (red‐NTR) with a fluorescence turn‐on response. This probe, a benzocoumarin dye functionalized with a (nitrobenzyl)pyridinium moiety, is stabilized through hydrogen bonding between its nitro group and the reduced cofactor flavin mononucleotide (FMNH2). This interaction suppresses both the enzymatic reduction and fluorescence quenching by photoinduced electron transfer. The probe selectively distinguishes red‐NTR from its oxidized form (ox‐NTR), allowing the observation of active enzyme levels in hypoxic cells, mouse tumor tissues, and cells undergoing premature senescence. The probe offers a unique and valuable tool for studying dynamic biological processes involving NTR under redox homeostasis. The first reversible binding probe (rNTRp) is presented that selectively detects the nitroreductase enzyme's active, reduced form (red‐NTR) with a fluorescence turn‐on response. Contrary to the conventional reaction‐based probes known to date, which provide only static information on the accumulated enzyme activity at a given time, rNTRp allows monitoring the active enzyme level in real time.

Bacterial nitroreductase enzymes capable of activating imaging probes and prodrugs are valuable tools for gene-directed enzyme prodrug therapies and targeted cell ablation models. We recently engineered a nitroreductase (E. coli NfsB F70A/F108Y) for the substantially enhanced reduction of the 5-nitroimidazole PET-capable probe, SN33623, which permits the theranostic imaging of vectors labeled with oxygen-insensitive bacterial nitroreductases. This mutant enzyme also shows improved activation of the DNA-alkylation prodrugs CB1954 and metronidazole. To elucidate the mechanism behind these enhancements, we resolved the crystal structure of the mutant enzyme to 1.98 Å and compared it to the wild-type enzyme. Structural analysis revealed an expanded substrate access channel and new hydrogen bonding interactions. Additionally, computational modeling of SN33623, CB1954, and metronidazole binding in the active sites of both the mutant and wild-type enzymes revealed key differences in substrate orientations and interactions, with improvements in activity being mirrored by reduced distances between the N5-H of isoalloxazine and the substrate nitro group oxygen in the mutant models. These findings deepen our understanding of nitroreductase substrate specificity and catalytic mechanisms and have potential implications for developing more effective theranostic imaging strategies in cancer treatment.

Developing precise targeted cell ablation in the axolotl ( Ambystoma mexicanum ) is crucial for elucidating the roles or interactions of specific cell types in regeneration and modeling diseases. Here we establish a Nitroreductase (NTR)-based inducible cell ablation system in axolotls. Through generation of Sox2:Cherry-NTR knock-in axolotls, we achieve efficient ablation of ependymoglial cells (EGCs) in the central nervous system. Combined spinal cord and brain transplantation and injury models demonstrate regeneration failure upon EGC depletion, suggesting that EGCs are sole source of central nervous system regeneration. Additionally, EGC ablation in the spinal cord resulted in delayed tail regeneration. Moreover, we establish NeuroD6:Cherry-NTR and NeuroD6:Cherry-NTR2.0 knock-in lines to ablate postmitotic cortical neurons, enable the investigation of brain regeneration after large-scale neuronal depletion. We found that NTR2.0 (but not NTR) leads to elimination of >95% of targeted neurons in the dorsal pallium. All lost neuronal subtypes are chronologically regenerated with laminar-like distribution mirroring developmental patterning. Finally, we create Cre-LoxP-based conditional NTR2.0 transgenic axolotls using a constitutive CAGGs promoter, enabling tissue-specific ablation of specific cell types when crossed to existing Cre lines. In summary, our study establishes an efficient and versatile targeted cell ablation system in axolotls, providing a valuable tool for deep dissection of tissue regeneration in axolotls. Here they establish an NTR-based genetic ablation tool in axolotls to investigate the function of specific cells. They use this system to demonstrate the capacity of ependymoglial cells for central nervous system regeneration.

Insight regarding how diverse enzymatic functions and reactions have evolved from ancestral scaffolds is fundamental to understanding chemical and evolutionary biology, and for the exploitation of enzymes for biotechnology. We undertook an extensive computational analysis using a unique and comprehensive combination of tools that include large-scale phylogenetic reconstruction to determine the sequence, structural, and functional relationships of the functionally diverse flavin mononucleotide-dependent nitroreductase (NTR) superfamily (>24,000 sequences from all domains of life, 54 structures, and >10 enzymatic functions). Our results suggest an evolutionary model in which contemporary subgroups of the superfamily have diverged in a radial manner from a minimal flavin-binding scaffold. We identified the structural design principle for this divergence: Insertions at key positions in the minimal scaffold that, combined with the fixation of key residues, have led to functional specialization. These results will aid future efforts to delineate the emergence of functional diversity in enzyme superfamilies, provide clues for functional inference for superfamily members of unknown function, and facilitate rational redesign of the NTR scaffold.