MOLECULAR BIOTECHNOLOGY
·
2016
·Vol. 58
·期 6
·428-440
·
IF 1.752
Yang Yuanyuan,Lin Jinping,Wei Dongzhi
A NADPH-dependent and FMN-containing nitroreductase (Gox0834) from Gluconobacter oxydans was cloned and heterogeneously expressed in Escherichia coli . The purified enzyme existed as a dimer with an apparent molecular mass of about 31.4 kDa. The enzyme displayed broad substrate specificity and reduced a variety of mononitrated, polynitrated, and polycyclic nitroaromatic compounds to the corresponding amino products. The highest activity was observed for the reduction of CB1954 (5-(1-aziridinyl)-2,4-dinitrobenzamide). The enzyme kinetics analysis showed that Gox0834 had relatively low K
m
(54 ± 11 μM) but high k
cat / K
m
value (0.020 s −1 /μM) for CB1954 when compared with known nitroreductases. Nitrobenzene and 2,4,6-trinitrotoluene (TNT) were preferred substrates for this enzyme with specific activity of 11.0 and 8.9 μmol/min/mg, respectively. Gox0834 exhibited a broad temperature optimum of 40–60 °C for the reduction of CB1954 with a pH optimum between 7.5 and 8.5. The purified enzyme was very stable below 37 °C over a broad pH range of 6.0–10.0. These characteristics suggest that the nitroreductase Gox0834 may be a possible candidate for catalyzing prodrug activation, bioremediation, or biocatalytic processes.
研究领域:
微生物学
·
酶学
·
环境科学
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产品关联
DOI
PubMed
高影响因子
JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC
·
2016
·
IF 2.189
Min Li,Zheng Zhou,Zhi-Jun Zhang,Hui-Lei Yu,Jian-He Xu
Nitrile reductase is a newly discovered enzyme class that can catalyze the reduction of nitriles directly to amines, a very important reaction in synthetic organic chemistry. However, little progress has been made towards the application of this biocatalysis reaction for organic synthesis since the initial discovery of nitrile reductase for over ten years. One of the potential reasons may be due to the lack of structural information and biochemical properties, in addition to its inherent narrow substrate scope. Meanwhile, the highly conserved active site residues make it a great challenge to widen the substrate spectrum of nitrile reductase by means of protein engineering approaches. To explore more nitrile reductases with different characteristics ( e.g . specific activity, thermostability, etc ) for potential synthetic applications, a new nitrile reductase, designated as Pc NRed, was discovered from the genome of Pectobacterium carotovorum , with a moderate specific activity of 366 U/g protein towards its natural substrate, preQ 0 . The Pc NRed was subsequently overexpressed in Escherichia coli BL21 (DE3), purified to homogeneity and then its biochemical properties were examined. The optimal reaction temperature and pH were determined to be 40 °C and 7.4, respectively. The thermal deactivation process of Pc NRed obeys first-order kinetics, with half-lives of 47.2, 13.6 and 3.9 h measured at 30, 40, and 50 °C, respectively. The kinetic constants were also determined with respect to both of the substrates NADPH and preQ 0 , giving k cat of 0.025 s −1 and 0.024 s −1 , and K M of 0.229 mM and <0.25 μM. To shed some light on the structural information of Pc NRed, a homology model was built and compared with its counterparts, which is helpful for further engineering of this novel but less-elucidated enzymes.
研究领域:
生物化学
·
酶工程
·
合成生物学
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产品关联
DOI
高影响因子
CHEMBIOCHEM
·
2017
·Vol. 19
·期 5
·521-526
·
IF 2.847
Zheng Zhou,Min Li,Jian-He Xu,Zhi-Jun Zhang
Graphical Reduced to clear : The nitrile reductase from Pectobacterium carotovorum is able to catalyze the reduction of nitriles to primary amines with the consumption of two molecules of NADPH. A single mutation at the entrance of the substrate channel increases both the activity and stability of the nitrile reductase. Nitrile reductases are considered to be promising and environmentally benign nitrile-reducing biocatalysts to replace traditional metal catalysts. Unfortunately, the catalytic efficiencies of the nitrile reductases reported so far are very low. To date, all attempts to increase the catalytic activity of nitrile reductases by protein engineering have failed. In this work, we successfully increased the specific activity of a nitrile reductase from Pectobacterium carotovorum from 354 to 526 U g prot −1 by engineering the substrate binding pocket; moreover, the thermostability was also improved (≈2-fold), showing half-lives of 140 and 32 h at 30 and 40 °C, respectively. In the bioreduction of 2-amino-5-cyanopyrrolo[2,3- d ]pyrimidin-4-one (preQ 0 ) to 2-amino-5-aminomethylpyrrolo[2,3- d ]pyrimidin-4-one (preQ 1 ), the variant was advantageous over the wild-type enzyme with a higher reaction rate and complete conversion of the substrate within a shorter period. Homology modeling and docking analysis revealed some possible origins of the increased activity and stability. These results establish a solid basis for future engineering of nitrile reductases to increase the catalytic efficiency further, which is a prerequisite for applying these novel biocatalysts in synthetic chemistry.
研究领域:
生物化学
·
合成生物学
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DOI
高影响因子
Advanced Healthcare Materials
·
2023
·
IF 10
Xianbao Sun,Lingling Xu,Hai-Dong Xu,Limin Xie,Rui Wang,Zhimou Yang,Wenjun Zhan,Shurong Shen,Gaolin Liang
Current molecular photoacoustic (PA) probes are designed with either stimulus-turned “on” or assembly-enhanced signals to trace biological analytes/events. PA probes based on the nature-derived click reaction between 2-cyano-6-aminobenzothiazole (CBT) and cysteine (Cys) (i.e., CBT-Cys click reaction) possess both “turn-on” and “enhanced” PA signals; and thus, should have higher sensitivity. Nevertheless, such PA probes, particularly those for sensitive imaging of tumor hypoxia, remain scarce. Herein, a PA probe NI-Cys(StBu)-Dap(IR780)-CBT (NI-C-CBT) is rationally designed, which after being internalized by hypoxic tumor cells, is cleaved by nitroreductase under the reduction condition to yield cyclic dimer C-CBT-Dimer to turn the PA signal “ON” and subsequently assembled into nanoparticles C-CBT-NPs with additionally enhanced PA signal (“Enhanced”). NI-C-CBT exhibits 1.7-fold “ON” and 3.2-fold overall “Enhanced” PA signals in vitro. Moreover, it provides 1.9-fold and 2.8-fold overall enhanced PA signals for tumor hypoxia imaging in HeLa cells and HeLa tumor-bearing mice, respectively. This strategy is expected to be widely applied to design more “smart” PA probes for sensitive imaging of important biological events in vivo in near future.
研究领域:
生物医学工程
·
分子影像学
·
肿瘤学
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DOI
PubMed
高影响因子
ANALYST
·
2018
·Vol. 144
·期 1
·284-289
·
IF 3.864
Lei Zhang,Xue Shan,Leilei Guo,Jikang Zhang,Junliang Ge,Qing Jiang,Xinghai Ning
Nitroreductase activities are positively associated with the hypoxic level of tumors, making it an attractive target for tumor detection. Herein, we have developed a 2,5-bis(methylsulfinyl)-1,4-diaminobenzene based probe (BBP), which is a nitroreductase (NTR) responsive fluorescent probe and can rapidly detect NTRs with high sensitivity and specificity. The BBP showed not only a selective response to NTRs over other biological reductants, but also high sensitivity to NTRs and could detect as low as 20 ng mL−1 NTRs. Furthermore, the BBP responded rapidly to NTRs in as fast as 10 minutes, enabling real-time monitoring of the production levels of NTRs. Most importantly, the BBP could identify NTR activities in 2D cell monolayers, 3D tumor spheroids, and even solid tumors in mice. Particularly, the BBP could monitor the early tumor formation and treatment response via measuring NTR activities. Overall, the BBP appears to be an ideal imaging probe for the detection of solid tumors, and possesses great potential in a broad range of diagnostic and therapeutic applications in the clinic.
研究领域:
肿瘤学
·
分子影像学
·
生物化学
✓ 已验证
DOI
PubMed
高影响因子
Analytical Methods
·
2021
·Vol. 13
·期 19
·2223-2228
·
IF 2.896
Bo-Wen Liu,Peng-Cheng Huang,Fang-Ying Wu
A novel and efficient light-controlled colorimetric assay for the quantification and detection of nitroreductase (NTR) was constructed based on p-aminophenol (pAP)-catalyzed and nicotinamide adenine dinucleotide (NADH)-mediated generation of AgNPs. Due to the hydrolysis of p-nitrophenol by NTR in the presence of NADH, the hydrolysis product can be used as a catalyst to catalyze the reduction of Ag+ by NADH under the light. As the concentration of NTR increases, the value of absorbance at ca. 400 nm (A400) decreases and the color of the solution turns from brown to bright yellow. A linear correlation was obtained between A400 and the NTR concentration in the range from 1–50 μg mL−1 and the limit of detection (LOD) is 0.27 μg mL−1. The detection system does not respond to other common biological molecules due to the specificity of enzymes and the effect of the nitroreductase inhibitor on the NTR activity was also tested. Finally, we applied the assay to determine NTR in human serum samples by spiking different concentrations of NTR with a recovery of 85.2%–92.5%.
研究领域:
生物化学
·
医学检测
·
纳米技术
✓ 已验证
DOI
PubMed
高影响因子
TALANTA
·
2021
·
IF 6.057
Bo-Wen Liu,Pengcheng Huang,Fang-Ying Wu
Hypoxia refers to the lack of oxygen supply to cells or tissues. The overexpression of nitroreductase has been shown to be closely related to the degree of hypoxia, which leads to the level of nitroreductase (NTR) being used as an indicator of hypoxia. We reported a facile visual detection of NTR based on the aggregation of gold and silver alloy nanoparticles . Compared with gold nanoparticles (AuNPs), the aggregation behavior of Au 80 Ag 20 NPs caused a more prominent color change. Copper ions (Cu 2+ ) can be rapidly reduced by nicotinamide adenine dinucleotide (NADH) under the catalysis of Au 80 Ag 20 NPs. But NADH is consumed as an electron donor during the catalytic reduction reaction of p-nitrophenol (pNP) by NTR. A decrease of NADH amount results in the aggregation of Au 80 Ag 20 NPs by the excess Cu 2+ and different aggregation degrees of Au 80 Ag 20 NPs lead to observable color change. A linear correlation of A 600 /A 505 = 0.0285 [NTR]+0.361 (R 2 = 0.980) was obtained with a limit of detection (LOD) of 0.23 μg/mL for UV–vis spectrophotometer. For visual detection, the values of R/B against the concentration of NTR obtains a calibration curve of R/B = −0.031 [NTR]+ 1.54 (R 2 = 0.985) with a LOD of 0.76 μg/mL, which is of the same order of magnitude as the UV–vis spectrophotometer analysis. As a comparison, Au 80 Ag 20 NPs was replaced by several different composition nanoparticles (Au NPs, Au 70 Ag 30 NPs, Au 50 Ag 50 NPs) to be a chromogenic substrate, and the results suggest the Au 80 Ag 20 NPs is the most sensitive substrate in our assay. Selectivity tests showed that the detection system did not respond to other common substances, and the reaction mechanism was verified by inhibitor research. Finally, the assay was used on the human serum samples with spiking NTR, and the recovery rates of this assay with UV–vis spectrophotometer were basically consistent with RGB analysis.
研究领域:
生物化学
·
医学检测
·
纳米技术
✓ 已验证
产品关联
DOI
PubMed
高影响因子
CHEMOSPHERE
·
2023
·
IF 8.8
Jie-wen Ma,Gui-wen Liu,Jia-Yu Zhai,Ke-qian Zhao,Ya-qing Wu,Rui-lian Yu,Gong-ren Hu,Yu Yan
Roxarsone (3-nitro-4-hydroxyphenylarsonic acid, Rox), a widely used organoarsenical feed additive, can enter soils and be further biotransformed into various arsenic species that pose human health and ecological risks. However, the pathway and molecular mechanism of Rox biotransformation by soil microbes are not well studied. Therefore, in this study, we isolated a Rox-transforming bacterium from manure-fertilized soil and identified it as Pseudomonas chlororaphis through morphological analysis and 16S rRNA gene sequencing. Pseudomonas chlororaphis was able to biotransform Rox to 3-amino-4-hydroxyphenylarsonic acid (3-AHPAA), N -acetyl-4-hydroxy- m -arsanilic acid (N-AHPAA), arsenate [As(V)], arsenite [As(III)], and dimethylarsenate [DMAs(V)]. The complete genome of Pseudomonas chlororaphis was sequenced. PcmdaB, encoding a nitroreductase, and PcnhoA, encoding an acetyltransferase, were identified in the genome of Pseudomonas chlororaphis . Expression of PcmdaB and PcnhoA in E. coli Rosetta was shown to confer Rox(III) and 3-AHPAA(III) resistance through Rox nitroreduction and 3-AHPAA acetylation , respectively. The PcMdaB and PcNhoA enzymes were further purified and functionally characterized in vitro . The kinetic data of both PcMdaB and PcNhoA were well fit to the Michaelis-Menten equation, and nitroreduction catalyzed by PcMdaB is the rate-limiting step for Rox transformation. Our results provide new insights into the environmental risk assessment and bioremediation of Rox(V)-contaminated soils.
研究领域:
环境微生物学
·
分子生物学
·
土壤科学
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DOI
PubMed
高影响因子
PLANT CELL AND ENVIRONMENT
·
2021
·Vol. 44
·期 9
·3173-3183
·
IF 7.228
Kunlong Su,Zhenying Wu,Yuchen Liu,Shanshan Jiang,Dongmei Ma,Yan Wang,Chunxiang Fu
Dinitrotoluene (DNT) has been extensively used in manufacturing munitions, polyurethane foams and other important chemical products. However, it is highly toxic and mutagenic to most organisms. Here, we synthesized a codon-optimized bacterial nitroreductase gene, NfsI, for plant expression. The kinetic analysis indicates that the recombinant NfsI can detoxify both 2,4-DNT and its sulfonate (DNTS), while it has a 97.6-fold higher catalytic efficiency for 2,4-DNT than DNTS. Furthermore, we overexpressed NfsI in switchgrass ( Panicum virgatum L.), which is a multiple-purpose crop used for fodder and biofuel production as well as phytoremediation. The 2,4-DNT treatment inhibited root elongation of wild-type switchgrass plants and promoted reactive oxygen species (ROS) accumulation in roots. In contrast, overexpression of NfsI in switchgrass significantly alleviated 2,4-DNT-induced root growth inhibition and ROS overproduction. Thus, the NfsI overexpressing transgenic switchgrass plant removed 94.1% 2,4-DNT after 6 days, whose efficiency was 1.7-fold higher than control plants. Moreover, the comparative transcriptome analysis suggests that 22.9% of differentially expressed genes induced by 2,4-DNT may participate in NfsI-mediated 2,4-DNT detoxification in switchgrass. Our work sheds light on the function of NfsI during DNT phytoremediation for the first time, revealing the application potential of switchgrass plants engineered with NfsI .
研究领域:
植物学
·
环境科学
·
遗传工程
✓ 已验证
DOI
高影响因子
CHINESE JOURNAL OF CHEMISTRY
·
2024
·
IF 5.5
Ximeng Shi,Yumeng Wang,Jiaoyang Yu,Yating Yang,Zecheng Jin,Hanghang Wang,Dalei Wu,Wei Chen,Jianming Guo,Yinan Zhang
Comprehensive Summary The detection of critical endogenous species, such as bacteria in microenvironments in the body, requires better imaging tools for visualization and monitoring of biological events. Bioluminescence imaging is the most popular strategy for obtaining real-time in living cells and organisms. Herein, we introduced a nitroaryl group on the C-3 position and a hydroxy group at the C-6 phenyl ring on furimazine to report the first bioluminescent probe ( 7 ) based on NanoLuc-furimazine bioluminescent pair for the detection of nitroreductase in bacteria. The probe, which possessed up to 560-fold intensity increase with a low detection limit of 16 ng/mL of nitroreductase, has the most efficient uncage efficiency in comparison with other bioluminescent congeners, thus enabling highly selective and sensitive visualization of NTR activity in a panel of clinical priority pathogens. Additionally, imaging of the recombinant strain as well as the NTR from mouse feces indicated the potential of this probe in the application of different mouse disease models.
研究领域:
生物化学
·
分子生物学
·
医学影像学
✓ 已验证
DOI
高影响因子
ANALYST
·
2014
·Vol. 140
·期 2
·574-581
·
IF 3.906
Jian Xu,Shaobo Sun,Qian Li,Ying Yue,Yingdong Li,Shijun Shao
A novel “Turn-On” fluorescent probe, quaternarized 4-pyridinyl-substituted BODIPY dye by incorporating a 5-nitrofuran moiety, was developed and applied for imaging the hypoxic status of tumor cells by the indirect detection of nitroreductase. The design was based on a nitroreductase-catalyzed reduction of the nitrofuran moiety in the presence of reduced nicotinamide adenine dinucleotide (NADH) as an electron donor and followed by the 1,6-rearrangement-elimination and the release of free 4-pyridinyl-substituted BODIPY dye 2. This probe displayed desired properties such as high specificity, “Turn-On” fluorescence response with suitable sensitivity, appreciable water solubility and rapid response time (within 5 min). Moreover, as a biocompatible molecule, the probe has been successfully applied for imaging the hypoxic status of tumor cells (e.g. A549 cells) and especially used for real-time determination of nitroreductase produced by Escherichia coli. Therefore, we hope to apply this novel method in the biomedical research fields for the imaging of disease-relevant hypoxia and detection of pathogenic microorganisms.
研究领域:
生物化学
·
医学影像
·
肿瘤学
✓ 已验证
DOI
PubMed
高影响因子
TALANTA
·
2019
·
IF 4.916
Lei Zhang,Leilei Guo,Xue Shan,Xiaohong Lin,Tingting Gu,Jikang Zhang,Junliang Ge,Wendi Li,Haixiong Ge,Qing Jiang,Xinghai Ning
Listeria monocytogenes ( Listeria ) is a facultative pathogenic bacterium, and a sensitive method for specific detection of Listeria is considered of clinically significant. However, current approaches for identifying Listeria are time consuming or tentative, and especially, cannot identify bacterial viability and antibiotic efficacy, which are critical in establishing a treatment recipe. Herein, we have developed a nitroreductase (NTR) responsive fluorescent probe (NRFP) with a fluorescence off-on feature, which could rapidly detect Listeria in vitro and in vivo with high specificity and sensitivity. NRFP showed a selective response to NTRs over other biological reductants, and could detect NTRs as low as 12.5 ng/mL. Furthermore, NRFP responded rapidly to NTRs within 10 min, enabling it real-time monitoring NTR production. Most importantly, NRFP could not only distinguish Listeria from other bacteria in vitro and in vivo for the first time, but could provide valuable information about Listeria desired for healthcare professionals, such as the presence and viability. Particularly, NRFP could real-time monitor antimicrobial effects in vivo , thereby identifying effective antibiotics for Listeria infections. Overall, NRFP appears to be an ideal imaging probe for Listeria , and possesses great potentials in diagnostic and therapeutic applications.
研究领域:
微生物学
·
医学影像
·
分子生物学
✓ 已验证
DOI
PubMed
高影响因子
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