Molecular Characterization and Substrate Specificity of Nitrobenzene Dioxygenase from Comamonas sp. Strain JS765

Abstract

Comamonas sp. strain JS765 can grow with nitrobenzene as the sole source of carbon, nitrogen, and energy. We report here the sequence of the genes encoding nitrobenzene dioxygenase (NBDO), which catalyzes the first step in the degradation of nitrobenzene by strain JS765. The components of NBDO were designated Reductase NBZ , Ferredoxin NBZ , Oxygenase NBZ␣ , and Oxygenase NBZ␤ , with the gene designations nbzAa, nbzAb, nbzAc, and nbzAd, respectively. Sequence analysis showed that the components of NBDO have a high level of homology with the naphthalene family of Rieske nonheme iron oxygenases, in particular, 2-nitrotoluene dioxygenase from Pseudomonas sp. strain JS42. The enzyme oxidizes a wide range of substrates, and relative reaction rates with partially purified Oxygenase NBZ revealed a preference for 3-nitrotoluene, which was shown to be a growth substrate for JS765. NBDO is the first member of the naphthalene family of Rieske nonheme iron oxygenases reported to oxidize all of the isomers of mono-and dinitrotoluenes with the concomitant release of nitrite.Nitroaromatic compounds are used extensively as industrial feedstocks for many manufacturing processes, including the production of pesticides, dyes, and explosives (11). Due to improper storage, use, and disposal, nitroaromatic compounds have been released into the environment, where they are considered environmental pollutants. For example, nitrobenzene and 2,4-and 2,6-dinitrotoluene are included in the U.S. Environmental Protection Agency's list of priority pollutants (14).The biodegradation of aromatic hydrocarbons and related compounds by aerobic bacteria is often initiated by multicomponent dioxygenase systems that catalyze the addition of both atoms of molecular oxygen to the substrate. Nitroaromatic compounds, in general, are resistant to oxidative attack due to the electron-withdrawing nature of the nitro groups and the stability of the benzene ring (29,33). Only recently have aerobic bacteria been isolated that utilize nitroaromatic compounds as growth substrates (19,32). One example is Comamonas sp. strain JS765, which can grow with nitrobenzene as the sole source of carbon, nitrogen, and energy. Previous experiments showed that JS765 uses an oxidative pathway for the degradation of nitrobenzene, with the initial reaction catalyzed by nitrobenzene 1,2-dioxygenase (NBDO; Fig. 1) (18). Other nitroarene dioxygenase genes from aerobic bacteria have been cloned and sequenced; these include genes encoding 2-nitrotoluene dioxygenase from Pseudomonas sp. strain JS42 (20) and 2,4-dinitrotoluene dioxygenases (DNTDOs) from Burkholderia sp. strain DNT (35) and Burkholderia cepacia R34 (G. R. Johnson, B. E. Haigler, R. K. Jain, and J. C. Spain, Abstr. 98th Gen. Meet. Am. Soc. Microbiol., abstr. Q-83, p. 435, 1998). However, strains DNT and R34 are unable to grow with nitrobenzene and we have observed only slight growth of JS42 with nitrobenzene.The majority of nitroaromatic compounds are synthetic, and therefore they have been present in the environment for...

Nitroaromatic Compounds, from Synthesis to Biodegradation

“…13D) is then used to complete metabolism of 3-methylcatechol to TCA cycle intermediates (62). Subsequent growth assays showed that JS42 is also able to grow on nitrobenzene (111). Cloning and sequencing of the genes encoding 2-nitrotoluene dioxygenase (2NTDO) revealed that it is a Rieske-type nonheme iron dioxygenase (later verified by biochemical analysis of the purified protein [146]) which has high sequence similarity to naphthalene dioxygenase from Pseudomonas sp.…”

“…13C). Biochemical and genetic analyses of nitrobenzene dioxygenase (NBDO) showed that it belongs to the naphthalene family of multicomponent Rieske-type dioxygenases (111). Crystal structures and site-directed mutagenesis studies of NBDO identified the active site of the enzyme, as well as key amino acids that bind and direct oxidation specifically to the nitrosubstituted carbon (48,91).…”

“…In addition to growing on nitrobenzene, Comamonas sp. strain JS765 is able to use 3-nitrotoluene as the sole carbon, nitrogen, and energy source for growth (111). NBDO oxidizes 3-nitrotoluene to 4-methylcatechol (Fig.…”

Effects of bioaugmentation in para-nitrophenol-contaminated soil on the abundance and community structure of ammonia-oxidizing bacteria and archaea

“…PNP was monitored at 280 nm with an Agilent ultraviolet detector, and its retention time was 11.2 min. The NH 4 + (Fawcett and Scott 1960) and NO 2 − (Lessner et al 2002) concentrations were colorimetrically detected (Chi et al 2013). The concentration of nitrate (NO 3 − ) was determined as described by Norman et al (1985).…”

Molecular Mechanisms of Nitroarene Degradation

“…Substrate specificity of naphthalene, 2-nitrotoluene and nitrobenzene dioxygenases. The genes for the nitrobenzene dioxygenase (NBDO) system and the 2-nitrotoluene dioxygenase (2NTDO) system have been cloned and sequenced (6,8) and the gene organization is shown in Fig. 5.…”