Gene Cloning, Purification, and Characterization of NfsB, a Minor Oxygen-Insensitive Nitroreductase from Escherichia coli, Similar in Biochemical Properties to FRase I, the Major Flavin Reductase in Vibrio fischeri

Zenno, Shuhei; Koike, Hideaki; Tanokura, Masaru; Saigo, Kaoru

doi:10.1093/oxfordjournals.jbchem.a021473

Cited by 150 publications

(165 citation statements)

References 42 publications

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“…It is a flavin reductase and can use either FAD or FMN as substrates, although the affinity for the latter is greater. NTR has flavin reductase activity, but the flavins are relatively poor substrates and reduction rates are low (G. M. Anlezark, unpublished data), as they are for a highly related protein NfsB, compared with proteins from other related species (Zenno et al, 1996). B. amyloliquefaciens YwrO is also an azoreductase, rapidly reducing the azodye o-methyl red.…”

Section: Discussionmentioning

confidence: 99%

Bacillus amyloliquefaciens orthologue of Bacillus subtilis ywrO encodes a nitroreductase enzyme which activates the prodrug CB 1954 The GenBank accession number for the sequence reported in this paper is AF373598.

Anlezark¹,

Vaughan²,

Fashola-Stone³

et al. 2002

Microbiology

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A nitroreductase with distinct properties that can activate the prodrug 5-aziridinyl-2,4-dinitrobenzamide (CB 1954) was isolated from Bacillus amyloliquefaciens. The encoding gene was identified as a homologue of the ywrO of Bacillus subtilis, and was obtained as a PCR product by reverse genetics, cloned and the entire nucleotide sequence determined. The gene was found to reside between homologues of the B. subtilis alsD and yswB genes ; however, the ywrO and yswB genes of B. amyloliquefaciens were not separated by a fourth gene, ywsA. The B. amyloliquefaciens ywrO gene was overexpressed, the recombinant protein purified and its properties were compared with those of two CB 1954-activating enzymes, Escherichia coli B nitroreductase (NTR) and Walker DT-diaphorase (DTD). In common with these enzymes menadione was an electron acceptor (K m 3 µM) and activity with this substrate was inhibited by the presence of dicoumarol (K i 1 0 µM).

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Section: Discussionmentioning

confidence: 99%

Bacillus amyloliquefaciens orthologue of Bacillus subtilis ywrO encodes a nitroreductase enzyme which activates the prodrug CB 1954 The GenBank accession number for the sequence reported in this paper is AF373598.

Anlezark¹,

Vaughan²,

Fashola-Stone³

et al. 2002

Microbiology

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“…NfsA includes the major nitroreductase in E. coli (3, 37) and Frp in V. harveyi (14,17). NfsB includes the minor nitroreductase in E. coli (3,20,38) and Frase I in Vibrio (Photobacterium) fischeri (6, 39).…”

Section: Cr(vi) To Cr(iii) Cr(iii) Was Much Less Toxic To E Coli Thmentioning

confidence: 99%

“…Type I is oxygen insensitive (3,6,15,17,20,37,38,39), and type II is oxygen sensitive (21, 26). In type I, there are two nitroflavin reductase superfamiles: NfsA and NfsB (3,6,14,17,20,37,38,39). NfsA includes the major nitroreductase in E. coli (3, 37) and Frp in V. harveyi (14,17).…”

mentioning

confidence: 99%

Vibrio harveyi Nitroreductase Is Also a Chromate Reductase

Kwak

Lee

Kim

2003

Appl Environ Microbiol

143

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The chromate reductase purified from Pseudomonas ambigua was found to be homologous with several nitroreductases. Escherichia coli DH5␣ and Vibrio harveyi KCTC 2720 nitroreductases were chosen for the present study, and their chromate-reducing activities were determined. A fusion between glutathione Stransferase (GST) and E. coli DH5␣ NfsA (GST-EcNfsA), a fusion between GST and E. coli DH5␣ NfsB (GST-EcNfsB), and a fusion between GST and V. harveyi KCTC 2720 NfsA (GST-VhNfsA) were prepared for their overproduction and easy purification. GST-EcNfsA, GST-EcNFsB, and GST-VhNFsA efficiently reduced nitrofurazone and 2,4,6-trinitrotoluene (TNT) as their nitro substrates. The K m values for GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA for chromate reduction were 11.8, 23.5, and 5.4 M, respectively. The V max values for GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA were 3.8, 3.9, and 10.7 nmol/min/mg of protein, respectively. GST-VhNfsA was the most effective of the three chromate reductases, as determined by each V max /K m value. The optimal temperatures of GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA for chromate reduction were 55, 30, and 30°C, respectively. Thus, it is confirmed that nitroreductase can also act as a chromate reductase. Nitroreductases may be used in chromate remediation. GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA have a molecular mass of 50 kDa and exist as a monomer in solution. Thin-layer chromatography showed that GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA contain FMN as a cofactor. GST-VhNfsA reduced Cr(VI) to Cr(III). Cr(III) was much less toxic to E. coli than Cr(VI).Environmental pollution by chromium may be severe. Chromium contamination is known to be prevalent at U.S. Department of Energy sites (29). The electroplating and leather-tanning industries also contribute to environmental contamination with Cr(VI) (23). Chromate compounds containing Cr(VI) are used widely in the cooling towers of heavy industry and atomic power plants, since Cr(VI) prevents corrosion and the growth of organisms (2). Cr(VI) is soluble, toxic, and carcinogenic, whereas Cr(III) is less soluble and less toxic (12). Thus, it is desirable to change Cr(VI) into Cr(III). This approach is taken in the bioremediation of Cr(VI) pollution. It shows promise for solving pollution problems and has advantages over various other physical and chemical methods.Chromate-reducing activities can be found in the cell extracts of many bacteria (4,5,8,10,13,18,25,30,(33)(34)(35). Chromate reductase can reduce the toxicity of Cr(VI) by reducing it to Cr(III) and lowering its solubility (5, 9). The chromate reductase from Pseudomonas ambigua has been purified and characterized (33). Chromate-reducing activities have been associated with DT-diaphorase (7) and aldehyde oxidase (1) in the cell cytoplasm. Cytochrome P450 located in the cell membrane is also known to have chromate-reducing activity (21). It seems that various reductases in the cell can function in chromate reduction. P. ambigua chromate reductase (33) has high homology with Escherichia coli NfsA (59%) ...

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“…The purified proteins were denatured with heat treatment at 11 70°C for 20 min to liberate the bound FMN, followed by centrifugation at 10,000 X g for 20 min [35]. 12 The supernatant was analysed by thin-layer chromatography (TLC) using a solvent system of Na2PO4 13 and the developed plates were visualized under UV light at 366 nm.…”

Section: Confirming the Presence Of Fmnmentioning

confidence: 99%

“…The yellow colour of the expressed protein solutions suggested that these 19 proteins were associated with FMN [35]. To prove the association with FMN, TLC analysis was 20 performed of denatured protein solutions and compared with a FMN and FAD standard (Figure 3).…”

Section: Presence Of Fmnmentioning

confidence: 99%

Identification of novel nitroreductases from Bacillus cereus and their interaction with the CB1954 prodrug

Gwenin

Poornima

Halliwell

et al. 2015

Biochemical Pharmacology

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This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Abstract 12Directed enzyme prodrug therapy is a form of cancer chemotherapy in which bacterial prodrug-13 activating enzymes, or their encoding genes, are directed to the tumour before administration of a 14 2 prodrug. The prodrug can then be activated into a toxic drug at the tumour site, reducing off-target 1 effects. The bacterial Nitroreductases are a class of enzymes used in this therapeutic approach and 2 although very promising, the low turnover rate of prodrug by the most studied nitroreductase enzyme, 3NfnB from E. coli (NfnB_Ec), is a major limit to this technology. There is a continual search for 4 enzymes with greater efficiency, and as part of the search for more efficient bacterial nitroreductase 5 enzymes, two novel enzymes from Bacillus cereus (strain ATCC 14579) have been identified and 6shown to reduce the CB1954 (5-(Aziridin-1-yl)-2,4-dinitrobenzamide) prodrug to its respective 2'-and 7 4'-hydroxylamine products. Both enzymes shared features characteristic of the Nitro-FMN-reductase 8Superfamily including non-covalently associated FMN, requirement for the NAD(P)H cofactor, 9 homodimeric, could be inhibited by Dicoumarol (3,3'-methylenebis(4-hydroxy-2H-chromen-2-one), 10 and displayed ping pong bi bi kinetics. Based on the biochemical characteristics and nucleotide 11 alignment with other nitroreductase enzymes, one enzyme was named YdgI_Bc and the other 12YfkO_Bc. Both B. cereus enzymes had greater turnover for the CB1954 prodrug compared with 13 NfnB_Ec, and in the presence of added NADPH cofactor, YfkO_Bc had superior cell killing ability, 14 and produced mainly the 4'-hydroxylamine product at low prodrug concentration. The YfkO_Bc was 15 identified as a promising candidate for future enzyme prodrug therapy. 16

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Gene Cloning, Purification, and Characterization of NfsB, a Minor Oxygen-Insensitive Nitroreductase from Escherichia coli, Similar in Biochemical Properties to FRase I, the Major Flavin Reductase in Vibrio fischeri

Cited by 150 publications

References 42 publications

Bacillus amyloliquefaciens orthologue of Bacillus subtilis ywrO encodes a nitroreductase enzyme which activates the prodrug CB 1954 The GenBank accession number for the sequence reported in this paper is AF373598.

Bacillus amyloliquefaciens orthologue of Bacillus subtilis ywrO encodes a nitroreductase enzyme which activates the prodrug CB 1954 The GenBank accession number for the sequence reported in this paper is AF373598.

Vibrio harveyi Nitroreductase Is Also a Chromate Reductase

Identification of novel nitroreductases from Bacillus cereus and their interaction with the CB1954 prodrug

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