Saturation mutagenesis of 2,4-DNT dioxygenase ofBurkholderia sp. strain DNT for enhanced dinitrotoluene degradation

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...

Section: Discussionsupporting

confidence: 64%

Section: Discussionmentioning

confidence: 99%

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

Parales

2006

“…The dark-brown color arises from the intracellular dihydroxylated product (in this case, hydroquinone) (Fig. 1), which auto-oxidizes to quinones upon secretion (17). DNA sequencing indicated there were two amino acid codon changes, M22L and L380I, in dntB.…”

Section: Resultsmentioning

confidence: 99%

“…This new variant, M22L/L380I, not only exhibits a wider range of oxygenation capabilities, it also maintains the original activity with small improvements for 4M5NC (1.4-fold) and 4NC (1.6-fold) degradation (Table 2). Our previous work using saturation mutagenesis had already enhanced 2,4-DNT dioxygenase activity (the first step in the 2,4-DNT degradation pathway) to create the DntAc variant I204L of strain DNT; this evolved enzyme degrades 2,4-DNT to 4M5NC twofold faster than wild-type 2,4-DNT dioxygenase, as well as having novel activity on 2,3-DNT and 2,5-DNT (17). Burkholderia sp.…”

Section: Discussionmentioning

confidence: 99%

Protein Engineering of the 4-Methyl-5-Nitrocatechol Monooxygenase from Burkholderia sp. Strain DNT for Enhanced Degradation of Nitroaromatics

Leungsakul

Johnson

Wood

2006

Self Cite

4-Methyl-5-nitrocatechol (4M5NC) monooxygenase (DntB) from Burkholderia sp. strain DNT catalyzes the second step of 2,4-dinitrotoluene degradation by converting 4M5NC to 2-hydroxy-5-methylquinone with the concomitant removal of the nitro group. DntB is a flavoprotein that has a very narrow substrate range. Here, error-prone PCR was used to create variant DntB M22L/L380I, which accepts the two new substrates 4-nitrophenol (4NP) and 3-methyl-4-nitrophenol (3M4NP). At 300 M of 4NP, the initial rate of the variant expressing M22L/L380I enzyme (39 ؎ 6 nmol/min/mg protein) was 10-fold higher than that of the wild-type enzyme (4 ؎ 2 nmol/min/mg protein). The values of k cat /K m of the purified wild-type DntB enzyme and purified variant M22L/L380I were 40 and 450 (s ؊1 M ؊1 ), respectively, which corroborates that the variant M22L/L380I enzyme has 11-fold-higher efficiency than the wild-type enzyme for 4NP degradation. In addition, the variant M22L/L380I enzyme has fourfold-higher activity toward 3M4NP; at 300 M, the initial nitrite release rate of M22L/L380I enzyme was 17 ؎ 4 nmol/min/mg protein, while that of the wild-type enzyme was 4.4 ؎ 0.7 nmol/min/mg protein. Saturation mutagenesis was also used to further investigate the role of the individual amino acid residues at positions M22, L380, and M22/L380 simultaneously. Mutagenesis at the individual positions M22L and L380I did not show appreciable enhancement in 4NP activity, which suggested that these two sites should be mutated together; simultaneous saturation mutagenesis led to the identification of the variant M22S/L380V, with 20% enhanced degradation of 4NP compared to the variant M22L/L380I. This is the first report of protein engineering for nitrite removal by a flavoprotein.

“…S2 in the supplemental material). Substitutions at the corresponding residue are known to affect the specificity of related enzymes, including naphthalene, toluene, and 2,4-dinitrotoluene dioxygenases (43)(44)(45). In NBDO, G204 interacts with the methyl group of 3NT when it is bound at the active site of the enzyme (42).…”

Section: Discussionmentioning

confidence: 99%

Selection for Growth on 3-Nitrotoluene by 2-Nitrotoluene-Utilizing Acidovorax sp. Strain JS42 Identifies Nitroarene Dioxygenases with Altered Specificities

Mahan

Penrod

Kass

et al. 2015

Acidovorax sp. strain JS42 uses 2-nitrotoluene as a sole source of carbon and energy. The first enzyme of the degradation pathway, 2-nitrotoluene 2,3-dioxygenase, adds both atoms of molecular oxygen to 2-nitrotoluene, forming nitrite and 3-methylcatechol. All three mononitrotoluene isomers serve as substrates for 2-nitrotoluene dioxygenase, but strain JS42 is unable to grow on 3-or 4-nitrotoluene. Using both long-and short-term selections, we obtained spontaneous mutants of strain JS42 that grew on 3-nitrotoluene. All of the strains obtained by short-term selection had mutations in the gene encoding the ␣ subunit of 2-nitrotoluene dioxygenase that changed isoleucine 204 at the active site to valine. Those strains obtained by long-term selections had mutations that changed the same residue to valine, alanine, or threonine or changed the alanine at position 405, which is just outside the active site, to glycine. All of these changes altered the regiospecificity of the enzymes with 3-nitrotoluene such that 4-methylcatechol was the primary product rather than 3-methylcatechol. Kinetic analyses indicated that the evolved enzymes had enhanced affinities for 3-nitrotoluene and were more catalytically efficient with 3-nitrotoluene than the wild-type enzyme. In contrast, the corresponding amino acid substitutions in the closely related enzyme nitrobenzene 1,2-dioxygenase were detrimental to enzyme activity. When cloned genes encoding the evolved dioxygenases were introduced into a JS42 mutant lacking a functional dioxygenase, the strains acquired the ability to grow on 3-nitrotoluene but with significantly longer doubling times than the evolved strains, suggesting that additional beneficial mutations occurred elsewhere in the genome. N itroaromatic compounds are toxic and stable in the environment and are important components in the manufacture of dyes, polymers, explosives, and pesticides (1). The synthesis and widespread use of these products have caused environmental contamination of soil and groundwater (2, 3). In addition, nitroaromatic compounds tend to undergo reduction, resulting in the formation of mutagenic aromatic amines (4). Due to the stability, toxicity, and mutagenicity of these chemicals, the U.S. Environmental Protection Agency has designated several nitroaromatic compounds as priority pollutants (5).Although the majority of nitroarene compounds are anthropogenic, a number of microorganisms have developed the ability to utilize compounds of this chemical class as sources of carbon, nitrogen, and energy (6). Bacterial strains capable of growth on nitrobenzene (7-9), mononitrotoluenes (10-16), and dinitrotoluenes (17-19) have been isolated from various contaminated environments. For example, Acidovorax sp. strain JS42 (formerly Pseudomonas sp. strain JS42) uses nitrobenzene (NB) and 2-nitrotoluene (2NT) as sole sources of carbon, nitrogen, and energy (15,20), while Comamonas sp. strain JS765 is capable of growth on NB and 3-nitrotoluene (3NT) (9, 21). The first enzymes of each degradation pathway, 2-nitro...