Purification and characterization of nitrobenzene nitroreductase from Pseudomonas pseudoalcaligenes JS45

Abstract

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-aminophenol 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 K m for nitrobenzene is 5 M at an initial NADPH concentration of 0.5 mM. The K m for NADPH at an initial nitrobenzene concentration of 0.1 mM is 183 M. 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 K m and the inducible control of enzyme synthesis suggest that nitrobenzene is the physiological substrate for this enzyme.

Characterization of a Pseudomonad 2-Nitrobenzoate Nitroreductase and Its Catabolic Pathway-Associated 2-Hydroxylaminobenzoate Mutase and a Chemoreceptor Involved in 2-Nitrobenzoate Chemotaxis

“…For the first time, a homology model of 2-NBA nitroreductase is available, which adds to our limited knowledge on the structure and function of nitroreductases in nitroaromatic metabolism for which a limited number of ge- (11,35,40,51,69). NbaA is a newly discovered representative of a family of homodimeric NADH:FMN oxidoreductase-like fold proteins that is different from the nitro/flavin reductase family.…”

Genetic and Structural Organization of the Aminophenol Catabolic Operon and Its Implication for Evolutionary Process

“…In the partial reductive pathway, NB is converted into 2-aminophenol (AP), which is subsequently metabolized via a meta-like cleavage pathway. Several enzymes involved in NB catabolism of Pseudomonas pseudoalcaligenes JS45 were purified and characterized (21,23,24,31,44). However, despite intensive studies on the catabolic pathway of NB and characterization of the relevant enzymes, a detailed molecular basis of and a regulatory mechanism for NB catabolism remain yet to be elucidated.…”

Assimilation of Nitrogen from Nitrite and Trinitrotoluene in Pseudomonas putida JLR11

“…A number of mono-and dinitroaromatic compounds have been shown to be mineralized by bacteria (8,9,11,12,21,26,27,29,30). However, no bacterium has been found to use 2,4,6-trinitrotoluene (TNT) as the sole carbon source, although some cases are known in which TNT can serve as a nitrogen source (1,2,4,6,27).…”