Purification, Characterization, and Crystallization of the Components of the Nitrobenzene and 2-Nitrotoluene Dioxygenase Enzyme Systems

Abstract

The protein components of the 2-nitrotoluene (2NT) and nitrobenzene dioxygenase enzyme systems from Acidovorax sp. strain JS42 and Comamonas sp. strain JS765, respectively, were purified and characterized. These enzymes catalyze the initial step in the degradation of 2-nitrotoluene and nitrobenzene. The identical shared reductase and ferredoxin components were monomers of 35 and 11.5 kDa, respectively. The reductase component contained 1. Nitroaromatic compounds are relatively rare in nature but are widely used in the chemical industry for the production of dyes, resins, pesticides, herbicides, explosives, and other useful materials such as polyurethane foams (27). Because these compounds have been introduced into the environment quite recently, bacteria have had little time to adapt to the presence of these new chemicals and evolve new pathways for their degradation. Numerous aerobic bacteria that are capable of utilizing specific nitroaromatic compounds as sole carbon, nitrogen, and energy sources have been isolated from contaminated soils and groundwaters (27). Acidovorax (formerly Pseudomonas [25]) sp. strain JS42 was isolated from a nitrobenzene-contaminated industrial facility in Mississippi by selection for growth on 2-nitrotoluene (2NT) (20). Comamonas sp. strain JS765, which was isolated from an industrial waste treatment plant in New Jersey, utilizes nitrobenzene as a sole carbon and nitrogen source (28). In each strain, only a single new enzymatic reaction is required to convert the nitroarene compound to an easily degraded, naturally occurring product. Nitrobenzene and 2NT degradation by Comamonas sp. strain JS765 and Acidovorax sp. strain JS42 is initiated by closely related multicomponent dioxygenase enzyme systems. The nitrobenzene dioxygenase system (NBDO) and 2-nitrotoluene dioxygenase system (2NTDO) add both atoms of molecular oxygen to the aromatic ring (1, 24). The proposed unstable nitrohydrodiol products rearrange, forming catechol or 3-methylcatechol with simultaneous release of nitrite (Fig. 1).NBDO and 2NTDO are members of the Rieske nonheme iron dioxygenase family, which includes the well-studied naphthalene dioxygenase enzyme system (NDO) (42). Enzymatic attack at the nitro-substituted carbon by NBDO and 2NTDO is critical for the elimination of nitrite, and this type of reaction has not been demonstrated for any of the related aromatic hydrocarbon dioxygenases. Each enzyme system is composed of three protein components: an iron-sulfur flavoprotein reductase, a Rieske [2Fe-2S] ferredoxin, and a Rieske [2Fe-2S] nonheme iron dioxygenase. The reductase and ferredoxin components of NBDO and 2NTDO transfer electrons from NADH to the oxygenase and are identical in sequence (24). Although the oxygenases from these enzyme systems have very similar deduced amino acid sequences, they have distinct but * Corresponding author. Section

Nitroaromatic Compounds, from Synthesis to Biodegradation

“…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. strain 9816-4 (143).…”

Structural Features in the KshA Terminal Oxygenase Protein That Determine Substrate Preference of 3-Ketosteroid 9 -Hydroxylase Enzymes

“…Characteristic for ROs, including KshA H37Rv , is the formation of trimers. These trimers either occur as ␣3 subunits (2,4,17,19) or may additionally include a smaller ␤ subunit of the oxygenase component, resulting in an (␣␤)3 enzyme complex (3,6,7,8,9,13,15,20). The active site of KshA H37Rv was shown to be composed of a ␤ sheet, including a loop region located at the entrance of the active site, flanked by two ␣ helices.…”

“…Several three-dimensional (3D) structures of ROs are currently available (3,4,6,7,8,9,13,15,17,19,20), including a single 3D structure of KshA, namely, that of M. tuberculosis H37Rv (Rv3526; KshA H37Rv ) (2). Although the protein sequences of ROs vary considerably, their tertiary structures are very similar overall.…”

Expression inEscherichia coliof Biphenyl 2,3-Dioxygenase Genes from a Gram-Positive Polychlorinated Biphenyl Degrader,Rhodococcus jostiiRHA1

“…Similarly, expression of the activity of hybrid ring-hydroxylating dioxygenase complexes constructed by the replacement of heterologous ferredoxin, 30) ferredoxin reductase, 12,[31][32][33][34] or ferredoxin plus ferredoxin reductase [34][35][36] components of heterologous origin has been reported. In some cases, replacement of the ferredoxin component with one of heterologous origin failed to yield activity, although replacement of the Containing RHA1 bphAc.…”

“…Introduction of recombinant plasmids into P. pseudoalcaligenes KF730 22) and R. erythropolis IAM1399 was done by electroporation. Cells were grown in 10 mL of LB medium at 30 C to OD 600 ¼ 1:0, harvested by centrifuging 10 min at 1;300 Â g, and washed 3 times with ice-cold 0.3 M sucrose. The cells were resuspended in ice-cold 0.5 M sucrose.…”