Active site residues controlling substrate specificity in 2-nitrotoluene dioxygenase from Acidovorax sp. strain JS42

Lee, Kyung-Seon; Parales, Juanito V.; Friemann, Rosmarie; Parales, Rebecca E.

doi:10.1007/s10295-005-0021-z

Cited by 28 publications

(29 citation statements)

References 39 publications

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“…Mutation of Asn258 to a Val in NBDO changed the regioselectivity of product formation (12). In Oxy, although the original and substituted residues are both hydrophobic, nevertheless, the mutation affected substrate specificity.…”

Section: Resultsmentioning

confidence: 99%

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Structural Basis of the Divergent Oxygenation Reactions Catalyzed by the Rieske Nonheme Iron Oxygenase Carbazole 1,9a-Dioxygenase

Inoue

Usami

Ashikawa

et al. 2014

Appl Environ Microbiol

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e Carbazole 1,9a-dioxygenase (CARDO), a Rieske nonheme iron oxygenase (RO), is a three-component system composed of a terminal oxygenase (Oxy), ferredoxin, and a ferredoxin reductase. Oxy has angular dioxygenation activity against carbazole. Previously, site-directed mutagenesis of the Oxy-encoding gene from Janthinobacterium sp. strain J3 generated the I262V, F275W, Q282N, and Q282Y Oxy derivatives, which showed oxygenation capabilities different from those of the wild-type enzyme. To understand the structural features resulting in the different oxidation reactions, we determined the crystal structures of the derivatives, both free and complexed with substrates. The I262V, F275W, and Q282Y derivatives catalyze the lateral dioxygenation of carbazole with higher yields than the wild type. A previous study determined the crystal structure of Oxy complexed with carbazole and revealed that the carbonyl oxygen of Gly178 hydrogen bonds with the imino nitrogen of carbazole. In these derivatives, the carbazole was rotated approximately 15, 25, and 25°, respectively, compared to the wild type, creating space for a water molecule, which hydrogen bonds with the carbonyl oxygen of Gly178 and the imino nitrogen of carbazole. In the crystal structure of the F275W derivative complexed with fluorene, C-9 of fluorene, which corresponds to the imino nitrogen of carbazole, was oriented close to the mutated residue Trp275, which is on the opposite side of the binding pocket from the carbonyl oxygen of Gly178. Our structural analyses demonstrate that the fine-tuning of hydrophobic residues on the surface of the substratebinding pocket in ROs causes a slight shift in the substrate-binding position that, in turn, favors specific oxygenation reactions toward various substrates. Rieske nonheme iron oxygenases (ROs) catalyze the initial oxygenation reaction of aromatic compounds (1). ROs are of interest for the biodegradation of aromatic pollutants and for synthetic applications requiring enantio-and regiospecific reactions. Some ROs break down toxic heterocyclic compounds by a single oxidization reaction. ROs are generally composed of two or three components, a terminal oxygenase (Oxy) and one or two electron transfer components. The Oxy is activated by the electron transfer component(s) and then catalyzes the oxygenation of the substrate. Recent biochemical and structural studies have revealed that Oxys typically exhibit an ␣ 3 or (␣␤) 3 configuration (2). The ␣ subunit contains a Rieske [2Fe-2S] cluster and a mononuclear iron. The Rieske [2Fe-2S] cluster accepts electrons from the electron transfer component(s) and transfers electrons to the mononuclear iron. The mononuclear iron at the active site activates molecular oxygen, which attacks the substrate. The two oxygen atoms of the molecular oxygen bind to tandemly linked carbon atoms in an aromatic ring, creating two hydroxyl groups in the cis configuration (3). This process is called lateral dioxygenation. The amino acid residues that affect the substrate specificity of ROs have bee...

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Section: Resultsmentioning

confidence: 99%

“…This process is called lateral dioxygenation. The amino acid residues that affect the substrate specificity of ROs have been identified (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). In these studies, the molecular mechanism by which substrate specificity is altered has been discussed on the basis of proposed structural similarities or molecular simulation.…”

mentioning

confidence: 99%

Structural Basis of the Divergent Oxygenation Reactions Catalyzed by the Rieske Nonheme Iron Oxygenase Carbazole 1,9a-Dioxygenase

Inoue

Usami

Ashikawa

et al. 2014

Appl Environ Microbiol

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“…The amino group of Asn-258 forms a hydrogen bond with the nitro groups of nitrobenzene and 3NT based on NBDO crystal structure data (6) and is required for efficient attack at the nitro-substituted carbon of nitroarene substrates in NBDO ( Fig. 2 and Table 2) and 2NTDO (18). Interestingly, DNTDOs do not have an asparagine at position 258 (Table 1).…”

Section: Discussionmentioning

confidence: 99%

“…This residue has been shown to be important for determining substrate specificity in several different dioxygenases (15,16,18,(30)(31)(32)47). Changes in product ratios formed by the I350F mutant can be attributed to the increased size of the residue.…”

Section: Discussionmentioning

confidence: 99%

Control of Substrate Specificity by Active-Site Residues in Nitrobenzene Dioxygenase

Parales

2006

Appl Environ Microbiol

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Nitrobenzene 1,2-dioxygenase from Comamonas sp. strain JS765 catalyzes the initial reaction in nitrobenzene degradation, forming catechol and nitrite. The enzyme also oxidizes the aromatic rings of mono-and dinitrotoluenes at the nitro-substituted carbon, but the basis for this specificity is not understood. In this study, site-directed mutagenesis was used to modify the active site of nitrobenzene dioxygenase, and the contribution of specific residues in controlling substrate specificity and enzyme performance was evaluated. The activities of six mutant enzymes indicated that the residues at positions 258, 293, and 350 in the ␣ subunit are important for determining regiospecificity with nitroarene substrates and enantiospecificity with naphthalene. The results provide an explanation for the characteristic specificity with nitroarene substrates. Based on the structure of nitrobenzene dioxygenase, substitution of valine for the asparagine at position 258 should eliminate a hydrogen bond between the substrate nitro group and the amino group of asparagine. Up to 99% of the mononitrotoluene oxidation products formed by the N258V mutant were nitrobenzyl alcohols rather than catechols, supporting the importance of this hydrogen bond in positioning substrates in the active site for ring oxidation. Similar results were obtained with an I350F mutant, where the formation of the hydrogen bond appeared to be prevented by steric interference. The specificity of enzymes with substitutions at position 293 varied depending on the residue present. Compared to the wild type, the F293Q mutant was 2.5 times faster at oxidizing 2,6-dinitrotoluene while retaining a similar K m for the substrate based on product formation rates and whole-cell kinetics.Nitroaromatic compounds are commonly used for the production of pesticides, dyes, and polymers (34). Widespread application and improper disposal of these chemicals have resulted in their release into the environment. Past synthesis of the explosive 2,4,6-trinitrotoluene at military facilities in the United States has contaminated soil and groundwater with mixtures of mono-, di-, and trinitrotoluene isomers (34, 39). Nitrotoluenes pose a significant risk to human health and the environment due to their acute toxicity and suspected carcinogenicity, and several of them are listed on the U.S. Environmental Protection Agency's list of priority pollutants (25,34,44). Additionally, nitroaromatic compounds are rapidly reduced to aromatic amines in the environment; these chemicals are established human carcinogens and piscicides (34). In general, nitroaromatic compounds are recalcitrant to biodegradation; however, bacterial strains that grow on and completely mineralize nitrobenzene, 2-nitrotoluene (2NT), 2,4-dinitrotoluene, or 2,6-dinitrotoluene have been isolated and characterized (8,13,23,24,40). In Comamonas sp. strain JS765, the key enzyme allowing growth on nitrobenzene is nitrobenzene 1,2-dioxygenase (NBDO), which catalyzes the oxidation of nitrobenzene to catechol and nitrite (24,29). Th...

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“…strain 9816-4 (143). Mutagenesis studies revealed that specificity is controlled by the C-terminal half of the dioxygenase (144), and like the case in NBDO (91), the asparagine at position 258 is critical for proper positioning of substrates in the active site for oxidative removal of the nitro group (107). A divergently transcribed LysR-type regulator activates transcription of the 2NTDO genes in response to nitroaromatic compounds (89,112).…”

Section: Pathways For Nitrotoluene Catabolismmentioning

confidence: 99%

Nitroaromatic Compounds, from Synthesis to Biodegradation

Parales

2010

Microbiol Mol Biol Rev

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741

470

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SUMMARY Nitroaromatic compounds are relatively rare in nature and have been introduced into the environment mainly by human activities. This important class of industrial chemicals is widely used in the synthesis of many diverse products, including dyes, polymers, pesticides, and explosives. Unfortunately, their extensive use has led to environmental contamination of soil and groundwater. The nitro group, which provides chemical and functional diversity in these molecules, also contributes to the recalcitrance of these compounds to biodegradation. The electron-withdrawing nature of the nitro group, in concert with the stability of the benzene ring, makes nitroaromatic compounds resistant to oxidative degradation. Recalcitrance is further compounded by their acute toxicity, mutagenicity, and easy reduction into carcinogenic aromatic amines. Nitroaromatic compounds are hazardous to human health and are registered on the U.S. Environmental Protection Agency's list of priority pollutants for environmental remediation. Although the majority of these compounds are synthetic in nature, microorganisms in contaminated environments have rapidly adapted to their presence by evolving new biodegradation pathways that take advantage of them as sources of carbon, nitrogen, and energy. This review provides an overview of the synthesis of both man-made and biogenic nitroaromatic compounds, the bacteria that have been identified to grow on and completely mineralize nitroaromatic compounds, and the pathways that are present in these strains. The possible evolutionary origins of the newly evolved pathways are also discussed.

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Active site residues controlling substrate specificity in 2-nitrotoluene dioxygenase from Acidovorax sp. strain JS42

Cited by 28 publications

References 39 publications

Structural Basis of the Divergent Oxygenation Reactions Catalyzed by the Rieske Nonheme Iron Oxygenase Carbazole 1,9a-Dioxygenase

Structural Basis of the Divergent Oxygenation Reactions Catalyzed by the Rieske Nonheme Iron Oxygenase Carbazole 1,9a-Dioxygenase

Control of Substrate Specificity by Active-Site Residues in Nitrobenzene Dioxygenase

Nitroaromatic Compounds, from Synthesis to Biodegradation

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