Applied Microbiology and Biotechnology

, Volume 76, Issue 6, pp 1271–1279 | Cite as

Azoreductase from Rhodobacter sphaeroides AS1.1737 is a flavodoxin that also functions as nitroreductase and flavin mononucleotide reductase

  • Guangfei LiuEmail author
  • Jiti Zhou
  • Hong Lv
  • Xuemin Xiang
  • Jing Wang
  • Mi Zhou
  • Yuanyuan Qv
Biotechnologically Relevant Enzymes and Proteins


Previously reported azoreductase (AZR) from Rhodobacter sphaeroides AS1.1737 was shown to be a flavodoxin possessing nitroreductase and flavin mononucleotide (FMN) reductase activities. The structure model of AZR constructed with SWISS-MODEL displayed a flavodoxin-like fold with a three-layer α/β/α structure. With nitrofurazone as substrate, the optimal pH value and temperature were 7.0 and 50°C, respectively. AZR could reduce a number of nitroaromatic compounds including 2,4-dinitrotoluene, 2,6-dinitrotoluene, 3,5-dinitroaniline, and 2,4,6-trinitrotoluene (TNT). TNT resulted to be the most efficient nitro substrate and was reduced to hydroxylamino-dinitrotoluene. Both NADH and NADPH could serve as electron donors of AZR, where the latter was preferred. Externally added FMN was also reduced by AZR via ping-pong mechanism and was a competitive inhibitor of NADPH, methyl red, and nitrofurazone. AZR with broad substrate specificity is a member of a new nitro/FMN reductase family demonstrating potential application in bioremediation.


  1. Agarwal R, Bonanno JB, Burley SK, Swaminathan S (2006) Structure determination of an FMN reductase from Pseudomonas aeruginosa PA01 using sulfur anomalous signal. Acta Crystallogr D 62:383–391CrossRefGoogle Scholar
  2. Barrows SE, Cramer CJ, Truhlar DG, Elovitz MS, Weber EJ (1996) Factors controlling region selectivity in the reduction of polynitroaromatics in aqueous solution. Environ Sci Technol 30:3028–3038CrossRefGoogle Scholar
  3. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  4. Bryant C, DeLuca M (1991) Purification and characterization of an oxygen-insensitive NAD(P)H nitroreductase from Enterobacter cloacae. J Biol Chem 266:4119–4125Google Scholar
  5. Caballero A, Ramos JL (2006) A double mutant of Pseudomonas putida JLR11 deficient in the synthesis of the nitroreductase PnrA and assimilatory nitrite reductase NasB is impaired for growth on 2,4,6-trinitrotoluene (TNT). Environ Microbiol 8:1306–1310CrossRefGoogle Scholar
  6. 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 7:1211–1219CrossRefGoogle Scholar
  7. Chen H, Hopper SL, Cerniglia CE (2005) Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH-dependent flavoprotein. Microbiology 151:1433–1441CrossRefGoogle Scholar
  8. Deller S, Sollner S, Trenker-El-Toukhy R, Jelesarov I, Gubitz GM, Macheroux P (2006) Characterization of a thermostable NADPH:FMN oxidoreductase from the mesophilic bacterium Bacillus subtilis. Biochemistry 45:7083–7091CrossRefGoogle Scholar
  9. DeMarini DM, Shelton ML, Bell DA (1996) Mutation spectra of chemical fractions of a complex mixture: role of nitroarenes in the mutagenic specificity of municipal waste incinerator emissions. Mutat Res 349:1–20Google Scholar
  10. Emery DD, Faessler PC (1997) First production-level bioremediation of explosives-contaminated soil in the United States. Ann N Y Acad Sci 829:326–340CrossRefGoogle Scholar
  11. Fiorella PD, Spain JC (1997) Transformation of 2,4,6-trinitrotoluene by Pseudomonas pseudoalcaligenes JS52. Appl Environ Microbiol 63:2007–2015Google Scholar
  12. Fiorenza S, Dunston KL, Ward CH (1991) Decision making—is bioremediation a viable option? J Hazard Mater 28:171–283CrossRefGoogle Scholar
  13. Fraaije MW, Mattevi A (2000) Flavoenzymes: diverse catalysts with recurrent features. Trends Biochem Sci 25:126–132CrossRefGoogle Scholar
  14. Green NK, Kerr DJ, Mautner V, Harris PA, Searle PF (2004) The nitroreductase/CB1954 enzyme-prodrug system. Methods Mol Med 90:459–477Google Scholar
  15. Groenewegen PEJ, de Bont JMA (1992) Degradation of 4-nitrobenzoate via 4-hydroxylaminobenzoate and 3,4-dihydroxybenzoate in Comamonas acidovorans NBA-10. Arch Microbiol 158:381–386CrossRefGoogle Scholar
  16. Hughes JB, Wang C, Yesland K, Bhadra R, Richardson A, Bennet G, Rudolph F (1998) Reduction of 2,4,6-trinitrotoluene by Clostridium acetobutylicum through hydroxylamino-nitrotoluene intermediates. Environ Toxicol Chem 17:343–348CrossRefGoogle Scholar
  17. Kadiyala V, Nadeau LJ, Spain JC (2003) Construction of Escherichia coli strains for conversion of nitroacetophenones to ortho-aminophenols. Appl Environ Microbiol 69:6520–6526CrossRefGoogle Scholar
  18. Kim HY, Song HG (2005) Purification and characterization of NAD(P)H-dependent nitroreductase I from Klebsiella sp. C1 and enzymatic transformation of 2,4,6-trinitrotoluene. Appl Microbiol Biotechnol 68:766–773CrossRefGoogle Scholar
  19. Kobori T, Sasaki H, Lee WC, Zenno S, Saigo K, Murphy ME, Tanokura M (2001) Structure and site-directed mutagenesis of a flavoprotein from Escherichia coli that reduces nitrocompounds: alteration of pyridine nucleotide binding by a single amino acid substitution. J Biol Chem 276:2816–2823CrossRefGoogle Scholar
  20. Kutty R, Bennett GN (2005) Biochemical characterization of trinitrotoluene transforming oxygen-insensitive nitroreductases from Clostridium acetobutylicum ATCC 824. Arch Microbiol 184:158–167CrossRefGoogle Scholar
  21. Kwak YH, Lee DS, Kim HB (2003) Vibrio harveyi nitroreductase is also a chromate reductase. Appl Environ Microbiol 69:4390–4395CrossRefGoogle Scholar
  22. Lenke H, Warrelmann J, Daun G, Hund K, Sieglen U, Walter U, Knackmuss HJ (1998) Biological treatment of TNT contaminated soil. 2. Biologically induced immobilization of the contaminants and full-scale application. Environ Sci Technol 32:1964–1971CrossRefGoogle Scholar
  23. Lewis TA, Ederer MM, Crawford RL, Crawford DL (1997) Microbial transformation of 2,4,6-trinitrotoluene. J Ind Microbiol Biotech 18:89–96CrossRefGoogle Scholar
  24. Liger D, Graille M, Zhou CZ, Leulliot N, Quevillon-Cheruel S, Blondeau K, Janin J, van Tilbeurgh H (2004) Crystal structure and functional characterization of yeast YLR011wp, an enzyme with NAD(P)H-FMN and ferric iron reductase activities. J Biol Chem 279:34890–34897CrossRefGoogle Scholar
  25. Liochev SI, Hausladen A, Fridovich I (1999) Nitroreductase A is regulated as a member of the soxRS regulon of Escherichia coli. Proc Natl Acad Sci U S A 96:3537–3539CrossRefGoogle Scholar
  26. Nishino SF, Spain JC (1993) Degradation of nitrobenzene by a Pseudomonas pseudoalcaligenes. Appl Environ Microbiol 59:2520–2525Google Scholar
  27. Nicholas KB, Nicholas HB Jr, Deerfield DW II (1997) GeneDoc: analysis and visualization of genetic variation. EMBnet News 4:1–4Google Scholar
  28. Nivinskas H, Koder RL, Anusevicius Z, Sarlauskas J, Miller AF, Cenas N (2001) Quantitative structure–activity relationships in two-electron reduction of nitroaromatic compounds by Enterobacter cloacae NAD(P)H:nitroreductase. Arch Biochem Biophys 385:170–178CrossRefGoogle Scholar
  29. Padda RS, Wang C, Hughes JB, Kutty R, Bennett GN (2003) Mutagenicity of nitroaromatic degradation compounds. Environ Toxicol Chem 22:2293–2297CrossRefGoogle Scholar
  30. Pak JW, Knoke KL, Noguera DR, Fox BG, Chambliss GH (2000) Transformation of 2,4,6-trinitrotoluene by purified xenobiotic reductase B from Pseudomonas fluorescens I-C. Appl Environ Microbiol 66:4742–4750CrossRefGoogle Scholar
  31. Paterson ES, Boucher SE, Lambert IB (2002) Regulation of the nfsA gene in Escherichia coli by SoxS. J Bacteriol 184:51–58CrossRefGoogle Scholar
  32. Peterson FJ, Manson RP, Hovsepian J, Holtzman JL (1979) Oxygen-sensitive and -insensitive nitroreduction by Escherichia coli and rat hepatic microsomes. J Biol Chem 254:4009–4014Google Scholar
  33. Riefler RG, Smets BF (2002) NAD(P)H:flavin mononucleotide oxidoreductase inactivation during 2,4,6-trinitrotoluene reduction. Appl Environ Microbiol 68:1690–1696CrossRefGoogle Scholar
  34. Sarlauskas J, Nemeikaite-Ceniene A, Anusevicius Z, Miseviciene L, Julvez MM, Medina M, Gomez-Moreno C, Cenas N (2004) Flavoenzyme-catalyzed redox cycling of hydroxylamino- and amino- metabolites of 2,4,6-trinitrotoluene: implications for their cytotoxicity. Arch Biochem Biophys 425:184–192CrossRefGoogle Scholar
  35. Schwede T, Kopp J, Guex N, Peitsch MC (2003) SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res 31:3381–3385CrossRefGoogle Scholar
  36. Searle PF, Chen MJ, Hu L, Race PR, Lovering AL, Grove JI, Guise C, Jaberipour M, James ND, Mautner V, Young LS, Kerr DJ, Mountain A, White SA, Hyde EI (2004) Nitroreductase: a prodrug-activating enzyme for cancer gene therapy. Clin Exp Pharmacol Physiol 31:811–816CrossRefGoogle Scholar
  37. Spain JC (1995) Biodegradation of nitroaromatic compounds. Annu Rev Microbiol 49:523–525CrossRefGoogle Scholar
  38. Suzuki Y, Yoda T, Ruhul A, Sugiura W (2001) Molecular cloning and characterization of the gene coding for azoreductase from Bacillus sp. OY1-2 isolated from soil. J Biol Chem 276:9059–9065CrossRefGoogle Scholar
  39. Tokiwa H, Ohnishi Y (1986) Mutagenicity and carcinogenicity of nitroarenes and their sources in the environment. CRC Crit Rev Toxicol 17:23–60Google Scholar
  40. Tsai TS (1991) Biotreatment of red water—a hazardous stream from explosive manufacture with fungal systems. Hazard Waste Hazard Mater 8:231–244Google Scholar
  41. Walker JE, Kaplan DL (1992) Biological degradation of explosives and chemical agents. Biodegradation 3:369–385CrossRefGoogle Scholar
  42. Watanabe M, Nishino T, Takio K, Sofuni T, Nohmi T (1998) Purification and characterization of wild-type and mutant “classical” nitroreductases of Salmonella typhimurium. L33R mutation greatly diminishes binding of FMN to the nitroreductase of S. typhimurium. J Biol Chem 273:23922–23928CrossRefGoogle Scholar
  43. Yan B, Zhou J, Wang J, Du C, Hou H, Song Z, Bao Y (2004) Expression and characteristics of the gene encoding azoreductase from Rhodobacter sphaeroides AS1.1737. FEMS Microbiol Lett 236:129–136CrossRefGoogle Scholar
  44. Zenno S, Koike H, Kumar AN, Jayaraman R, Tanokura M, Saigo K (1996a) 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 178:4508–4514Google Scholar
  45. Zenno S, Koike H, Tanokura M, Saigo K (1996b) 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 reductases in Vibrio fischeri. J Biochem 120:736–744Google Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Guangfei Liu
    • 1
    Email author
  • Jiti Zhou
    • 1
  • Hong Lv
    • 1
  • Xuemin Xiang
    • 1
  • Jing Wang
    • 1
  • Mi Zhou
    • 1
  • Yuanyuan Qv
    • 1
  1. 1.School of Environmental and Biological Science and TechnologyDalian University of TechnologyDalianPeople’s Republic of China

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