This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Somerville, C. C. Articles by Spain, J. C. Search for Related Content PubMed PubMed Citation Articles by Somerville, C. C. Articles by Spain, J. C.

 Previous Article  |  Next Article 

J. Bacteriol., 07 1995, 3837-3842, Vol 177, No. 13
Copyright © 1995, American Society for Microbiology

Purification and characterization of nitrobenzene nitroreductase from Pseudomonas pseudoalcaligenes JS45

CC Somerville, SF Nishino and JC Spain
Armstrong Laboratory, Tyndall Air Force Base, Florida 32403-5323, USA.

Pseudomonas pseudoalcaligenes JS45 grows on nitrobenzene as a sole source of carbon, nitrogen, and energy. The catabolic pathway involves reduction to hydroxylaminobenzene followed by rearrangement to o-amino- phenol and ring fission (S. F. Nishino and J. C. Spain, Appl. Environ. Microbiol. 59:2520, 1993). A nitrobenzene-inducible, oxygen-insensitive nitroreductase was purified from extracts of JS45 by ammonium sulfate precipitation followed by anion-exchange and gel filtration chromatography. A single 33-kDa polypeptide was detected by denaturing gel electrophoresis. The size of the native protein was estimated to be 30 kDa by gel filtration. The enzyme is a flavoprotein with a tightly bound flavin mononucleotide cofactor in a ratio of 2 mol of flavin per mol of protein. The Km for nitrobenzene is 5 microM at an initial NADPH concentration of 0.5 mM. The Km for NADPH at an initial nitrobenzene concentration of 0.1 mM is 183 microM. Nitrosobenzene was not detected as an intermediate of nitrobenzene reduction, but nitrosobenzene is a substrate for the enzyme, and the specific activity for nitrosobenzene is higher than that for nitrobenzene. These results suggest that nitrosobenzene is formed but is immediately reduced to hydroxylaminobenzene. Hydroxylaminobenzene was the only product detected after incubation of the purified enzyme with nitrobenzene and NADPH. Hydroxylaminobenzene does not serve as a substrate for further reduction by this enzyme. The products and intermediates are consistent with two two-electron reductions of the parent compound. Furthermore, the low Km and the inducible control of enzyme synthesis suggest that nitrobenzene is the physiological substrate for this enzyme.

This article has been cited by other articles:

• Ma, Y.-F., Wu, J.-F., Wang, S.-Y., Jiang, C.-Y., Zhang, Y., Qi, S.-W., Liu, L., Zhao, G.-P., Liu, S.-J. (2007). Nucleotide Sequence of Plasmid pCNB1 from Comamonas Strain CNB-1 Reveals Novel Genetic Organization and Evolution for 4-Chloronitrobenzene Degradation. Appl. Environ. Microbiol. 73: 4477-4483 [Abstract] [Full Text]  
• Iwaki, H., Muraki, T., Ishihara, S., Hasegawa, Y., Rankin, K. N., Sulea, T., Boyd, J., Lau, P. C. K. (2007). Characterization of a Pseudomonad 2-Nitrobenzoate Nitroreductase and Its Catabolic Pathway-Associated 2-Hydroxylaminobenzoate Mutase and a Chemoreceptor Involved in 2-Nitrobenzoate Chemotaxis. J. Bacteriol. 189: 3502-3514 [Abstract] [Full Text]  
• Wu, J.-f., Jiang, C.-y., Wang, B.-j., Ma, Y.-f., Liu, Z.-p., Liu, S.-j. (2006). Novel Partial Reductive Pathway for 4-Chloronitrobenzene and Nitrobenzene Degradation in Comamonas sp. Strain CNB-1.. Appl. Environ. Microbiol. 72: 1759-1765 [Abstract] [Full Text]  
• Caballero, A., Esteve-Nunez, A., Zylstra, G. J., Ramos, J. L. (2005). Assimilation of Nitrogen from Nitrite and Trinitrotoluene in Pseudomonas putida JLR11. J. Bacteriol. 187: 396-399 [Abstract] [Full Text]  
• Kadiyala, V., Nadeau, L. J., Spain, J. C. (2003). Construction of Escherichia coli Strains for Conversion of Nitroacetophenones to ortho-Aminophenols. Appl. Environ. Microbiol. 69: 6520-6526 [Abstract] [Full Text]  
• Riefler, R. G., Smets, B. F. (2002). NAD(P)H:Flavin Mononucleotide Oxidoreductase Inactivation during 2,4,6-Trinitrotoluene Reduction. Appl. Environ. Microbiol. 68: 1690-1696 [Abstract] [Full Text]  
• Esteve-Nunez, A., Caballero, A., Ramos, J. L. (2001). Biological Degradation of 2,4,6-Trinitrotoluene. Microbiol. Mol. Biol. Rev. 65: 335-352 [Abstract] [Full Text]  
• Park, H.-S., Kim, H.-S. (2001). Genetic and Structural Organization of the Aminophenol Catabolic Operon and Its Implication for Evolutionary Process. J. Bacteriol. 183: 5074-5081 [Abstract] [Full Text]  
• Davis, J. K., Paoli, G. C., He, Z., Nadeau, L. J., Somerville, C. C., Spain, J. C. (2000). Sequence Analysis and Initial Characterization of Two Isozymes of Hydroxylaminobenzene Mutase from Pseudomonas pseudoalcaligenes JS45. Appl. Environ. Microbiol. 66: 2965-2971 [Abstract] [Full Text]  
• Hawari, J., Halasz, A., Sheremata, T., Beaudet, S., Groom, C., Paquet, L., Rhofir, C., Ampleman, G., Thiboutot, S. (2000). Characterization of Metabolites during Biodegradation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine (RDX) with Municipal Anaerobic Sludge. Appl. Environ. Microbiol. 66: 2652-2657 [Abstract] [Full Text]  
• Park, H.-S., Kim, H.-S. (2000). Identification and Characterization of the Nitrobenzene Catabolic Plasmids pNB1 and pNB2 in Pseudomonas putida HS12. J. Bacteriol. 182: 573-580 [Abstract] [Full Text]  
• Schenzle, A., Lenke, H., Spain, J. C., Knackmuss, H.-J. (1999). Chemoselective Nitro Group Reduction and Reductive Dechlorination Initiate Degradation of 2-Chloro-5-Nitrophenol by Ralstonia eutropha JMP134. Appl. Environ. Microbiol. 65: 2317-2323 [Abstract] [Full Text]  
• Katsivela, E., Wray, V., Pieper, D. H., Wittich, R.-M. (1999). Initial Reactions in the Biodegradation of 1-Chloro-4-Nitrobenzene by a Newly Isolated Bacterium, Strain LW1. Appl. Environ. Microbiol. 65: 1405-1412 [Abstract] [Full Text]