Substrate Selectivity of a 3-Nitrophenol-Induced Metabolic System in Pseudomonas putida 2NP8 Transforming Nitroaromatic Compounds into Ammonia under Aerobic Conditions

Abstract

The 3-nitrophenol-induced enzyme system in cells of Pseudomonas putida 2NP8 manifested a wide substrate range in transforming nitroaromatic compounds through to ammonia production. All of the 30 mono-or dinitroaromatic substrates except 4-nitrophenol, 2,4-dinitrophenol, 2,4,6-trinitrophenol, 3-nitroaniline, 2-nitrobenzoic acid, and 2-nitrofuran were quickly transformed. Ammonia production from most nitroaromatic substrates appeared to be stoichiometric.The 3-nitrophenol (NP)-induced enzyme pathway in Pseudomonas putida 2NP8 is a nitroreductase-initiated metabolic system (12, 13). This system converted nitrobenzene (NB), a cometabolic substrate, into aminophenol, with subsequent hydrolysis into ammonia and benzoquinone ( Fig. 1A; RϭH). We postulated that this strain converted the growth substrate 3-NP into hydroxylquinol via oxidation of aminohydroquinone into imine, hydrolysis of the imine into quinone, and reduction of the quinone (13). Many reports describe hydroxylquinol as an intermediate in bacterial metabolism of a wide range of aromatic compounds (4, 8, 10). Here we describe the substrate selectivity of the 3-NP-induced metabolic system.Transformation of NAs by 3-NP-grown cells. We used a previously reported procedure (11-13) to test transformation of 30 nitroaromatic compounds (NAs) by 3-NP-grown cells of P. putida 2NP8. Initial transformation rates (Table 1) of most of the NAs were close to the transformation rates of 3-NP and NB. Loss of substrates in controls with killed cells was negligible for all NAs and 2-nitrofuran, except for 1-nitronaphthalene, 50% of which was retained by biomass within a 3-h incubation period.Transformation rates were greatly affected by hydroxyl groups located at the 2-or 4-position relative to the nitro group or by a carboxylic group at the 2-position only. Cells showed good transformation ability toward all of the position-4-substituted NAs, including dinitrotoluenes, except where that substituent was a hydroxyl group. The cells also quickly transformed all of the NAs with substitutions, other than hydroxyl or carboxyl groups, at the 2-position relative to the nitro group. The presence of a hydroxyl group at the 2-and 4-position relative to the nitro group reduced the transformation rate. We observed low or no transformation activity toward 4-NP, 2,4-dinitrophenol, 2,4,6-trinitrophenol, and 2-nitrobenzoic acid. 2-NP had a lower transformation rate than 3-NP. Substitutions at the 3-position did not reduce the transformation rate.It was noteworthy that 1-nitronaphthalene was also quickly removed. 2-Nitrofuran was transformed at a relatively low rate, indicating that the initial nitroreductase of this 3-NP-induced enzyme system (12) may be different from the nonspecific nitrofuran nitroreductase found in Escherichia coli (1, 2). Uninduced cells transformed only nitrobenzyl alcohol and nitrobenzaldehyde. Glucose-grown cells (8 mg of wet cells/ml [1.5 h]) exhibited little biotransformation activity toward all of the 30 NAs except for 2-or 4-nitrobenzaldehyde and 3-or

Isolation and characterization of an efficient nitro-reducing bacterium, Streptomyces mirabils DUT001, from soil

“…It has been reported that the majority of these bacteria could metabolite nitroaromatic compounds through reductive pathways (MarvinSikkema and de Bont 1994). Besides, bacterium often exhibits substrate selectivity and favors certain position of substituent group (Zhao and Ward 2001). In this study, Streptomyces mirabils DUT001 exhibited high activity to reduce nitro polycyclic aromatic compounds including 4-nitro-1, 8-naphthalic anhydride (4-NNA), 3-nitro-1, 8-naphthalic anhydride, 4-nitrophthalimide, 3-nitrophthalimide, as well as polynitrated aromatic compounds including 1, 4-dinitrobenzene and 1, 3-dinitrobenzene.…”

“…However, the reduction efficiency varies upon substrate structure and substituent position (Zhao and Ward 2001), making the application of microorganisms in biodegradation of nitroaromatic pollutants still challenging.…”

2-Nitrobenzoate 2-Nitroreductase (NbaA) Switches Its Substrate Specificity from 2-Nitrobenzoic Acid to 2,4-Dinitrobenzoic Acid under Oxidizing Conditions

“…Iwaki and colleagues (3) showed that Asn40, Asp76, and Glu113 in the conserved region of NbaA are necessary for binding to a divalent metal ion implicated in FMN binding, and that an insertion loop of 10 amino acids at positions 65 to 74 mediates NADPH binding. It exhibits a narrow specificity toward 2-NBA, which is different from other nitro/ flavin reductases (8)(9)(10). However, the catalytic properties and substrate specificity of NbaA have not been characterized.…”

Interactions among mononitrophenol isomers during biodegradation of their mixtures

“…The rationale for monitoring these nitrogen-containing products was a well-established paradigm that 3-NP metabolism appears to follow a different path as far as the ultimate form of nitrogen produced is concerned. Namely, it was shown earlier that the nitro-group of 3-NP must undergo reduction to ammonia [27][28][29] rather than a release as nitrite, as for the other NPs. [30,31] No release of nitrate was also observed in our previous work when 3-NP was used as the sole carbon and nitrogen source.…”

“…Members of the Pseudomonas genus were reported to transform a wide range of substrates, [39] including 30 mono-and di-nitroaromatic compounds, [29] via rather diversified metabolic pathways. Such a significant metabolic versatility may be linked to the way of genetic encoding of the enzymes participating in the degradation pathways.…”