Abstract
A kinetic comparison between three nitroreductase enzymes isolated from the genome of Bacillus licheniformis ATCC 14580 for prospective use as immobilised enzymes for explosives detection has been conducted. The genes encoding the three enzymes (yfkO [BLNfnB] encoding an NfsB-like enzyme; nfrA [BLNfrA1] and ycnD [BLNfrA2] encoding PnrA-like enzymes) have been PCR amplified from the native genome and cloned into pET-28a(+) and a modified cysteine(6)-tagged pET-28a(+) and subsequently over-expressed, purified, and biochemically characterised. The previously uncharacterised nitroreductases exhibited activity against a wide range of explosives, including cyclic nitramines. Amino acid alignments and overall structural comparisons with other nitroreductase family members suggest that the B. licheniformis enzymes are members of the NfsA-Frp/NfsB-FRase I family group. Despite the overall low amino acid identity, regions for flavin mononucleotide binding and active site residues were highly conserved.
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References
Berne C, Betancor L, Luckarift HR, Spain JC (2006) Application of a microfluidic reactor for screening cancer prodrug activation using silica-immobilized nitrobenzene nitroreductase. Biomacromolecules 7:2631–2636
Blehert DS, Fox BG, Chambliss GH (1999) Cloning and sequence analysis of two Pseudomonas flavoprotein xenobiotic reductases. J Bacteriol 181:6254–6263
Bryant C, DeLuca M (1991) Purification and characterization of an oxygen-insensitive NAD(P)H nitroreductase from Enterobacter cloacae. J Biol Chem 266:4119–4125
Bryant DW, McCalla DR, Leeksma M, Laneuville P (1981) Type I nitroreductases of Escherichia coli. Can J Microbiol 27:81–86
Bryant C, Hubbard L, McElroy WD (1991) Cloning, nucleotide sequence, and expression of the nitroreductase gene from Enterobacter cloacae. J Biol Chem 266:4126–4130
Caballero A, Lazaro JJ, Ramos JL, Esteve-Nunez A (2005) PnrA a new nitroreductase-family enzyme in the TNT-degrading strain Pseudomonas putida JLR11. Environ Microbiol 7(8):1211–1219
Gonzalez-Perez M, van Dillewijn MP, Wittich RM, Ramos JL (2007) Escherichia coli has multiple enzymes that attack TNT and release nitrogen for growth. Environ Microbiol 9:1535–1540
Gwenin CD, Kalaji M, Williams PA, Jones RM (2007) The orientationally controlled assembly of genetically modified enzymes in an amperometric biosensor. Biosens Bioelectron 22:2869–2875
Gwenin CD, Kalaji M, Kay CM, Williams PA, Tito DN (2008) An in situ amperometric biosensor for the detection of vapours from explosive compounds. Analyst 133:621–625
Kobori T, Sasaki H, Lee WC, Zenno S, Saigo K, Murphy MEP, Tanokura M (2001) Structure and site-directed mutagenesis of a flavoprotein from Escherichia coli that reduces nitrocompounds. Alteration of pyrimidine nucleotide binding by a single amino acid substitution. J Biol Chem 276:2816–2823
Larkin M, Blackshields AG, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Sequence analysis. Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948
Lee B-U, Park S-C, Cho Y-S, Kahng H-Y, Oh K-H (2008) Expression and characterization of the TNT nitroreductase of Pseudomonas sp. HK-6 in Escherichia coli. Curr Microbiol 56:386–390
Parkinson GN, Skelly JV, Neidle S (2000) Crystal structure of FMN-dependent nitroreductase from Escherichia coli B: a prodrug activating enzyme. J Med Chem 43:3624–3631
Peterson FJ, Mason RP, Hovsepian J, Holtzman JL (1979) Oxygen-sensitive and -insensitive nitroreduction by Escherichia coli and rat hepatic microsomes. J Biol Chem 254:4009–4014
Rey MW, Ramaiya P, Nelson BA, Brody-Karpin SD, Zaretsky EJ, Tang M, de Leon AL, Xiang H, Gusti V, Clausen IG, Olsen PB, Rasmussen MD, Andersen JT, Jorgensen PL, Larsen TS, Sorokin A, Bolotin A, Lapidus A, Galleron N, Ehrlich SD, Berka RM (2004) Complete genome of the industrial bacterium Bacillus licheniformis and comparisons with closely related Bacillus species. Genome Biol 5:R77
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, New York
Seth-Smith HMB, Rosser SJ, Basran A, Travis ER, Dabs ER, Nicklin S, Bruce NC (2002) Cloning, sequencing and characterization of the hexahydro-1,3,5-trinitro-1,3,5-triazine degradation gene cluster from Rhodococcus rhodochrous. Appl Environ Microbiol 68:4764–4771
Watanabe M, Ishidate M Jr, Nohmi T (1990) Nucleotide sequence of Salmonella typhimurimum nitroreductase gene. Nucleic Acids Res 18:1059
Whiteway J, Koziarz P, Veall J, Sandhu N, Kumar P, Hoecher B, Lambert IB (1998) Oxygen-insensitive nitroreductases: analysis of the roles of nfsA and nfsB in development of resistance to 5-nitrofuran derivatives in Escherichia coli. J Bacteriol 180:5529–5539
Williams PA, Zaba BN (1997) EnzPack for Windows. Biosoft, Cambridge, UK
Zenno S, Saigo K, Kanoh H, Inouye S (1994) Identification of the gene encoding the major NAD(P)H-flavin oxidoreductase of the bioluminescent bacteria Vibrio fischeri ATCC 7744. J Bacteriol 176:3536–3543
Zenno S, Koike H, Tanokura M, Saigo K (1996a) 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. J Biochem 120:736–744
Zenno S, Koike H, Kumar AN, Jayaraman R, Tanokura M, Saigo K (1996b) Biochemical characterization of NfsA, the Escherichia coli major nitroreductase exhibiting a high amino acid sequence homology to Frp, a Vibrio harveyi flavin oxidoreductase. J Bacteriol 178:4508–4514
Zenno S, Koike H, Tanokura M, Saigo K (1996c) Conversion of NfsB, a minor Escherichia coli nitroreductase, to a flavin reductase similar in biochemical properties to FRase I, the major flavin reductase in Vibrio fischeri, by a single amino acid substitution. J Bacteriol 178:4731–4733
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The authors wish to acknowledge funding from Trwyn and Nanosecure an EU project.
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Gwenin, C.D., Kalaji, M., Williams, P.A. et al. A kinetic analysis of three modified novel nitroreductases. Biodegradation 22, 463–474 (2011). https://doi.org/10.1007/s10532-010-9418-0
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DOI: https://doi.org/10.1007/s10532-010-9418-0