Skip to main content
SpringerLink
Log in

Heterologous Overexpression and Biochemical Characterization of a Nitroreductase from Gluconobacter oxydans 621H

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Roldán, M. D., Pérez-Reinado, E., Castillo, F., & Moreno-Vivián, C. (2008). Reduction of polynitroaromatic compounds: The bacterial nitroreductases. FEMS Microbiology Reviews, 32, 474–500.

    Article  Google Scholar 

  2. Misal, S., Bajoria, V., Lingojwar, D., & Gawai, K. (2013). Purification and characterization of nitroreductase from red alkaliphilic bacterium Aquiflexum sp. DL6. Applied Biochemistry and Microbiology, 49, 227–232.

    Article  CAS  Google Scholar 

  3. Green, L. K., Storey, M. A., Williams, E. M., Patterson, A. V., Smaill, J. B., Copp, J. N., & Ackerley, D. F. (2013). The flavin reductase MsuE is a novel nitroreductase that can efficiently activate two promising next-generation prodrugs for gene-directed enzyme prodrug therapy. Cancers, 5, 985–997.

    Article  CAS  Google Scholar 

  4. Xie, B., Yang, J., & Yang, Q. (2010). Isolation and characterization of an efficient nitro-reducing bacterium, Streptomyces mirabilis DUT001, from soil. World Journal of Microbiology and Biotechnology, 26, 855–862.

    Article  CAS  Google Scholar 

  5. Bryant, C., & Deluca, M. (1991). Purification and characterization of an oxygen-insensitive NAD (P) H nitroreductase from Enterobacter cloacae. Journal of Biological Chemistry, 266, 4119–4125.

    CAS  Google Scholar 

  6. Singh, S. N. (2014). Biological remediation of explosive residues. Cham: Springer.

    Book  Google Scholar 

  7. Naal, Z., Park, J.-H., Bernhard, S., Shapleigh, J., Batt, C., & Abruna, H. (2002). Amperometric TNT biosensor based on the oriented immobilization of a nitroreductase maltose binding protein fusion. Analytical Chemistry, 74, 140–148.

    Article  CAS  Google Scholar 

  8. Chaignon, P., Cortial, S., Ventura, A., Lopes, P., Halgand, F., Laprevote, O., & Ouazzani, J. (2006). Purification and identification of a Bacillus nitroreductase: Potential use in 3,5-DNBTF biosensoring system. Enzyme and Microbial Technology, 39, 1499–1506.

    Article  CAS  Google Scholar 

  9. Nguyen-Tran, H.-H., Zheng, G.-W., Qian, X.-H., & Xu, J.-H. (2014). Highly selective and controllable synthesis of arylhydroxylamines by the reduction of nitroarenes with an electron-withdrawing group using a new nitroreductase Ba NTR1. Chemical Communications, 50, 2861–2864.

    Article  CAS  Google Scholar 

  10. Nadeau, L., He, Z., & Spain, J. (2000). Production of 2-amino-5-phenoxyphenol from 4-nitrobiphenyl ether using nitrobenzene nitroreductase and hydroxylaminobenzene mutase from Pseudomonas pseudoalcaligenes JS45. Journal of Industrial Microbiology and Biotechnology, 24, 301–305.

    Article  CAS  Google Scholar 

  11. Xu, G., & McLeod, H. L. (2001). Strategies for enzyme/prodrug cancer therapy. Clinical Cancer Research, 7, 3314–3324.

    CAS  Google Scholar 

  12. Sekar, T. V., Foygel, K., Ilovich, O., & Paulmurugan, R. (2014). Noninvasive theranostic imaging of HSV1-sr39TK-NTR/GCV-CB1954 dual-prodrug therapy in metastatic lung lesions of MDA-MB-231 triple negative breast cancer in mice. Theranostics, 4, 460.

    Article  CAS  Google Scholar 

  13. Palmer, D. H., Mautner, V., Mirza, D., Oliff, S., Gerritsen, W., van der Sijp, J. R., et al. (2004). Virus-directed enzyme prodrug therapy: Intratumoral administration of a replication-deficient adenovirus encoding nitroreductase to patients with resectable liver cancer. Journal of Clinical Oncology, 22, 1546–1552.

    Article  CAS  Google Scholar 

  14. Zhang, J., Kale, V., & Chen, M. (2015). Gene-directed enzyme prodrug therapy. The AAPS Journal, 17, 102–110.

    Article  CAS  Google Scholar 

  15. Prosser, G., Copp, J., Syddall, S., Williams, E., Smaill, J., Wilson, W., et al. (2010). Discovery and evaluation of Escherichia coli nitroreductases that activate the anti-cancer prodrug CB1954. Biochemical Pharmacology, 79, 678–687.

    Article  CAS  Google Scholar 

  16. Voak, A. A., Gobalakrishnapillai, V., Seifert, K., Balczo, E., Hu, L., Hall, B. S., & Wilkinson, S. R. (2013). An essential type I nitroreductase from Leishmania major can be used to activate leishmanicidal prodrugs. Journal of Biological Chemistry, 288, 28466–28476.

    Article  CAS  Google Scholar 

  17. Vass, S., Jarrom, D., Wilson, W., Hyde, E., & Searle, P. (2009). E. coli NfsA: An alternative nitroreductase for prodrug activation gene therapy in combination with CB1954. British Journal of Cancer, 100, 1903–1911.

    Article  CAS  Google Scholar 

  18. Emptage, C. D., Knox, R. J., Danson, M. J., & Hough, D. W. (2009). Nitroreductase from Bacillus licheniformis: A stable enzyme for prodrug activation. Biochemical Pharmacology, 77, 21–29.

    Article  CAS  Google Scholar 

  19. Barak, Y., Thorne, S. H., Ackerley, D. F., Lynch, S. V., Contag, C. H., & Matin, A. (2006). New enzyme for reductive cancer chemotherapy, YieF, and its improvement by directed evolution. Molecular Cancer Therapeutics, 5, 97–103.

    Article  CAS  Google Scholar 

  20. Anlezark, G. M., Vaughan, T., Fashola-Stone, E., Michael, N. P., Murdoch, H., Sims, M. A., et al. (2002). Bacillus amyloliquefaciens orthologue of Bacillus subtilis ywrO encodes a nitroreductase enzyme which activates the prodrug CB 1954. Microbiology, 148, 297–306.

    Article  CAS  Google Scholar 

  21. Berne, C., Betancor, L., Luckarift, H. R., & Spain, J. C. (2006). Application of a microfluidic reactor for screening cancer prodrug activation using silica-immobilized nitrobenzene nitroreductase. Biomacromolecules, 7, 2631–2636.

    Article  CAS  Google Scholar 

  22. Swe, P., Copp, J., Green, L., Guise, C., Mowday, A., Smaill, J., et al. (2012). Targeted mutagenesis of the Vibrio fischeri flavin reductase FRase I to improve activation of the anticancer prodrug CB1954. Biochemical Pharmacology, 84, 775–783.

    Article  CAS  Google Scholar 

  23. Liu, G., Zhou, J., Lv, H., Xiang, X., Wang, J., Zhou, M., & Qv, Y. (2007). Azoreductase from Rhodobacter sphaeroides AS1. 1737 is a flavodoxin that also functions as nitroreductase and flavin mononucleotide reductase. Applied Microbiology and Biotechnology, 76, 1271–1279.

    Article  CAS  Google Scholar 

  24. Greenberg, W., Weiner, D., Adger, B., & Burk, M. (2003) High throughput screening of nitroreductases and use of the enzyme for biocatalytic reduction of nitro compounds and production of amines. WO Patent.

  25. Sheng, B., Xu, J., Zhang, Y., Jiang, T., Deng, S., Kong, J., et al. (2015). Utilization of d-lactate as an energy source supports the growth of Gluconobacter oxydans. Applied and Environmental Microbiology, 81, 4098–4110.

    Article  CAS  Google Scholar 

  26. Chu, H., Xin, B., Liu, P., Wang, Y., Li, L., Liu, X., et al. (2015). Metabolic engineering of Escherichia coli for production of (2S, 3S)-butane-2, 3-diol from glucose. Biotechnology for Biofuels, 8, 1–11.

    Article  Google Scholar 

  27. Gupta, A., Singh, V. K., Qazi, G., & Kumar, A. (2001). Gluconobacter oxydans: its biotechnological applications. Journal of Molecular Microbiology and Biotechnology, 3, 445–456.

    CAS  Google Scholar 

  28. Prust, C., Hoffmeister, M., Liesegang, H., Wiezer, A., Fricke, W. F., Ehrenreich, A., et al. (2005). Complete genome sequence of the acetic acid bacterium Gluconobacter oxydans. Nature Biotechnology, 23, 195–200.

    Article  CAS  Google Scholar 

  29. Zenno, S., Koike, H., Kumar, A. N., Jayaraman, R., Tanokura, M., & Saigo, K. (1996). Biochemical characterization of NfsA, the Escherichia coli major nitroreductase exhibiting a high amino acid sequence homology to Frp, a Vibrio harveyi flavin oxidoreductase. Journal of Bacteriology, 178, 4508–4514.

    CAS  Google Scholar 

  30. Kim, H.-Y., & Song, H.-G. (2005). Purification and characterization of NAD (P) H-dependent nitroreductase I from Klebsiella sp. C1 and enzymatic transformation of 2,4,6-trinitrotoluene. Applied Microbiology and Biotechnology, 68, 766–773.

    Article  Google Scholar 

  31. Liu, X., Chen, R., Yang, Z., Wang, J., Lin, J., & Wei, D. (2014). Characterization of a putative stereoselective oxidoreductase from gluconobacter oxydans and its application in producing ethyl (R)-4-chloro-3-hydroxybutanoate ester. Molecular Biotechnology, 56, 285–295.

    Article  CAS  Google Scholar 

  32. Daeid, N. N., Savage, K. A., Ramsay, D., Holland, C., & Sutcliffe, O. B. (2014). Development of gas chromatography–mass spectrometry (GC–MS) and other rapid screening methods for the analysis of 16 ‘legal high’ cathinone derivatives. Science and Justice, 54, 22–31.

    Article  Google Scholar 

  33. Chandor, A., Dijols, S., Ramassamy, B., Frapart, Y., Mansuy, D., Stuehr, D., et al. (2008). Metabolic activation of the antitumor drug 5-(Aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) by NO synthases. Chemical Research in Toxicology, 21, 836–843.

    Article  CAS  Google Scholar 

  34. Adams, M. A., & Jia, Z. (2006). Modulator of drug activity B from Escherichia coli: crystal structure of a prokaryotic homologue of DT-diaphorase. Journal of Molecular Biology, 359, 455–465.

    Article  CAS  Google Scholar 

  35. Koder, R. L., & Miller, A. F. (1998). Steady-state kinetic mechanism, stereospecificity, substrate and inhibitor specificity of Enterobacter cloacae nitroreductase. Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology, 1387, 395–405.

    Article  CAS  Google Scholar 

  36. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.

    Article  CAS  Google Scholar 

  37. Gwenin, C. D., Kalaji, M., Williams, P. A., & Kay, C. M. (2011). A kinetic analysis of three modified novel nitroreductases. Biodegradation, 22, 463–474.

    Article  CAS  Google Scholar 

  38. Morokutti, A., Lyskowski, A., Sollner, S., Pointner, E., Fitzpatrick, T. B., Kratky, C., et al. (2005). Structure and function of YcnD from Bacillus subtilis, a flavin-containing oxidoreductase. Biochemistry, 44, 13724–13733.

    Article  CAS  Google Scholar 

  39. Gwenin, V. V., Poornima, P., Halliwell, J., Ball, P., Robinson, G., & Gwenin, C. D. (2015). Identification of novel nitroreductases from Bacillus cereus and their interaction with the CB1954 prodrug. Biochemical Pharmacology, 98, 392–402.

    Article  CAS  Google Scholar 

  40. Iwaki, H., Muraki, T., Ishihara, S., Hasegawa, Y., Rankin, K. N., Sulea, T., et al. (2007). Characterization of a pseudomonad 2-nitrobenzoate nitroreductase and its catabolic pathway-associated 2-hydroxylaminobenzoate mutase and a chemoreceptor involved in 2-nitrobenzoate chemotaxis. Journal of Bacteriology, 189, 3502–3514.

    Article  CAS  Google Scholar 

  41. Yin, Y., Xiao, Y., Liu, H.-Z., Hao, F., Rayner, S., Tang, H., & Zhou, N.-Y. (2010). Characterization of catabolic meta-nitrophenol nitroreductase from Cupriavidus necator JMP134. Applied Microbiology and Biotechnology, 87, 2077–2085.

    Article  CAS  Google Scholar 

  42. Kutty, R., & Bennett, G. N. (2005). Biochemical characterization of trinitrotoluene transforming oxygen-insensitive nitroreductases from Clostridium acetobutylicum ATCC 824. Archives of Microbiology, 184, 158–167.

    Article  CAS  Google Scholar 

  43. Shin, J.-H., & Song, H.-G. (2009). Nitroreductase II involved in 2,4,6-trinitrotoluene degradation: Purification and characterization from Klebsiella sp. Cl. The Journal of Microbiology, 47, 536–541.

    Article  CAS  Google Scholar 

  44. French, C. E., Nicklin, S., & Bruce, N. C. (1998). Aerobic degradation of 2,4,6-trinitrotoluene by Enterobacter cloacae PB2 and by pentaerythritol tetranitrate reductase. Applied and Environmental Microbiology, 64, 2864–2868.

    CAS  Google Scholar 

  45. Pak, J. W., Knoke, K. L., Noguera, D. R., Fox, B. G., & Chambliss, G. H. (2000). Transformation of 2,4,6-Trinitrotoluene by Purified Xenobiotic Reductase B from Pseudomonas fluorescens I-C. Applied and Environmental Microbiology, 66, 4742–4750.

    Article  CAS  Google Scholar 

  46. Zhang, X., Zhang, B., Lin, J., & Wei, D. (2015). Oxidation of ethylene glycol to glycolaldehyde using a highly selective alcohol dehydrogenase from Gluconobacter oxydans. Journal of Molecular Catalysis. B, Enzymatic, 112, 69–75.

    Article  CAS  Google Scholar 

  47. Çelik, A., & Yetiş, G. (2012). An unusually cold active nitroreductase for prodrug activations. Bioorganic and medicinal chemistry, 20, 3540–3550.

    Article  Google Scholar 

Download references

Acknowledgments

This work was financially supported by the National Key Basic Research Development Program of China (“973” Program, No. 2012CB721003), the Natural Science Foundation of China (No. 21276084), Shanghai Natural Science Foundation (No. 15ZR1408600), and the National Major Science and Technology Projects of China (No. 2012ZX09304009).

Author information

Authors and Affiliations

  1. State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, People’s Republic of China

    Yuanyuan Yang, Jinping Lin & Dongzhi Wei

Authors
  1. Yuanyuan Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jinping Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dongzhi Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jinping Lin.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 353 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yang, Y., Lin, J. & Wei, D. Heterologous Overexpression and Biochemical Characterization of a Nitroreductase from Gluconobacter oxydans 621H. Mol Biotechnol 58, 428–440 (2016). https://doi.org/10.1007/s12033-016-9942-1

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-016-9942-1

Keywords

Search

Navigation