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

2,4-Dinitrotoluene (2,4-DNT) and 2,6-DNT are priority pollutants, and 2,4-DNT dioxygenase of Burkholderia sp. strain DNT (DDO) catalyzes the initial oxidation of 2,4-DNT to form 4-methyl-5-nitrocatechol and nitrite but has significantly less activity on other dinitrotoluenes and nitrotoluenes (NT). Hence, oxidation of 2,3-DNT, 2,4-DNT, 2,5-DNT, 2,6-DNT, 2NT, and 4NT were enhanced here by performing saturation mutagenesis on codon I204 of the alpha subunit (DntAc) of DDO and by using a membrane agar plate assay to detect catechol formation. Rates of degradation were quantified both by the formation of nitrite and by the formation of the intermediates with high performance liquid chromatography. The degradation of both 2,3-DNT and 2,5-DNT were achieved for the first time (no detectable activity with the wild-type enzyme) using whole Escherichia coli TG1 cells expressing DDO variants DntAc I204L and I204Y (0.70 +/- 0.03 and 0.22 +/- 0.02 nmol/min/mg protein for 2,5-DNT transformation, respectively). DDO DntAc variant I204L also transformed both 2,6-DNT and 2,4-DNT 2-fold faster than wild-type DDO (0.8 +/- 0.6 nmol/min/mg protein and 4.7 +/- 0.5 nmol/min/mg protein, respectively). Moreover, the activities of DDO for 2NT and 4NT were also enhanced 3.5-fold and 8-fold, respectively. Further, DntAc variant I204Y was also discovered with comparable rate enhancements for the substrates 2,4-DNT, 2,6-DNT, and 2NT but not 4NT. Sequencing information obtained during this study indicated that the 2,4-DNT dioxygenases of Burkholderia sp. strain DNT and B. cepacia R34 are more closely related than originally reported. This is the first report of engineering an enzyme for enhanced degradation of nitroaromatic compounds and the first report of degrading 2,5-DNT.     

Tendency of using different aromatic compounds as substrates by 2,4-DNT dioxygenase expressed by pJS39 carrying the gene dntA from Burkholderia sp. strain DNT

The substrate range of 2,4-dinitrotoluene (DNT) dioxygenase was investigated by measuring substrate-dependent O₂ uptake and maximum growth (expressed in A₆₀₀) on substrate-containing minimal medium. The control for each strain had no added particular substrate. The following aromatic compounds: catechol, α-naphthalene acetic acid, β-dimethylaminobenzaldehyde, 3,4-dinitrosalicylic acid, p-nitrophenol, naphthanol, o-anisic acid, salicylic acid, toluene, and benzoic acid, were tried as possible substrates. Considering all substrates used, only p-nitrophenol showed zero oxygen uptake rate and zero growth. This indicates that it was rather unlikely that p-nitrophenol is a substrate analog for 2,4-DNT. Catechol was clearly used as a sole carbon source by both wild-type Escherichia. coli (JM103) and the dnt transformant (JS39). Using α-naphthalene acetic acid and β-dimethylaminobenzaldehyde as substrates resulted in DNT dioxygenase oxygen uptake rates of 11.8 and 14?μM/hr/mg protein, respectively. However, using both compounds as a carbon source, JS39 had twice the growth rate of E. coli JM103. For the remaining six substrates tested (3, 4-dinitrosalicylic acid, p-nitrophenol, o-anisic acid, salicylic acid, toluene, and benzoic acid), there appeared to be growth advantages for JS39 (even though the growth in the presence of substrate was less than the controls) suggesting a situation similar to that described for α-naphthalene and β-dimethylaminobenzaldehyde above. Combining results from our assay with respirometry and growth-based experiments will allow a better understanding of the biochemical consequences of these interactions. These results suggest that DNT dioxygenase gene, dntA carried by JS39, and those potential genes for substrates-degraded enzyme(s) system could have a common root.     

Quantitative 1H-NMR analysis reveals steric and electronic effects on the substrate specificity of benzoate dioxygenase in Ralstonia eutropha B9

The cis-dihydroxylation of arenes by Rieske dearomatizing dioxygenases (RDDs) represents a powerful tool for the production of chiral precursors in organic synthesis. Here, the substrate specificity of the RDD benzoate dioxygenase (BZDO) in Ralstonia eutropha B9 whole cells was explored using quantitative ¹H nuclear magnetic resonance spectroscopy (q¹H-NMR). The specific activity, specific carbon uptake, and regioselectivity of the dihydroxylation reaction were evaluated in resting cell cultures for a panel of 17 monosubstituted benzoates. Two new substrates of this dioxygenase system were identified (2-methyl- and 3-methoxybenzoic acid) and the corresponding cis-diol metabolites were characterized. Higher activities were observed for benzoates with smaller substituents, predominantly at the 3-position. Elevated activities were also observed in substrates bearing greater partial charge at the C-2 position of the benzoate ring. The regioselectivity of the reaction was directly measured using q¹H-NMR and found to have positive correlation with increasing substituent size. These results widen the pool of cis-diol metabolites available for synthetic applications and offer a window into the substrate traits that govern specificity for BZDO.     

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

Acidovorax (formerly Pseudomonas) sp. strain JS42 utilizes 2-nitrotoluene as sole carbon, nitrogen, and energy source. 2-Nitrotoluene 2,3-dioxygenase (2NTDO) catalyzes the initial step in 2-nitrotoluene degradation by converting 2-nitrotoluene to 3-methylcatechol. In this study, we identified specific amino acids at the active site that control specificity. The residue at position 350 was found to be critical in determining both the enantiospecificity of 2NTDO with naphthalene and the ability to oxidize the ring of mononitrotoluenes. Substitution of Ile350 by phenylalanine resulted in an enzyme that produced 97% (+)-(1R, 2S)-cis-naphthalene dihydrodiol, in contrast to the wild type, which produced 72% (+)-(1R, 2S)-cis-naphthalene dihydrodiol. This substitution also severely reduced the ability of the enzyme to produce methylcatechols from nitrotoluenes. Instead, the methyl group of each nitrotoluene isomer was preferentially oxidized to form the corresponding nitrobenzyl alcohol. Substitution of a valine at position 258 significantly changed the enantiospecificity of 2NTDO (54% (−)-(1S, 2R)-cis-naphthalene dihydrodiol formed from naphthalene) and the ability of the enzyme to oxidize the aromatic ring of nitrotoluenes. Based on active site modeling using the crystal structure of nitrobenzene 1,2 dioxygenase from Comamonas sp. JS765, Asn258 appears to contribute to substrate specificity through hydrogen bonding to the nitro group of nitrotoluenes.     

Episodic positive selection during the evolution of naphthalene dioxygenase to nitroarene dioxygenase

Using different maximum-likelihood models of adaptive evolution, signatures of natural selective pressure, operating across the naphthalene family of dioxygenases, were examined. A lineage- and branch-site specific combined analysis revealed that purifying selection pressure dominated the evolutionary history of the enzyme family. Specifically, episodic positive Darwinian selection pressure, affecting only a few sites in a subset of lineages, was found to be responsible for the evolution of nitroarene dioxygenases (NArDO) from naphthalene dioxygenase (NDO). Site-specific analysis confirmed the absence of diversifying selection pressure at any particular site. Different sets of positively selected residues, obtained from branch-site specific analysis, were detected for the evolution of each NArDO. They were mainly located around the active site, the catalytic pocket and their adjacent regions, when mapped onto the 3D structure of the α-subunit of NDO. The present analysis enriches the current understanding of adaptive evolution and also broadens the scope for rational alteration of substrate specificity of enzyme by directed evolution.     

Directed evolution of aldolases for exploitation in synthetic organic chemistry

This review focuses on the directed evolution of aldolases with synthetically useful properties. Directed evolution has been used to address a number of limitations associated with the use of wild-type aldolases as catalysts in synthetic organic chemistry. The generation of aldolase enzymes with a modified or expanded substrate repertoire is described. Particular emphasis is placed on the directed evolution of aldolases with modified stereochemical properties: such enzymes can be useful catalysts in the stereoselective synthesis of biologically active small molecules. The review also describes some of the fundamental insights into mechanistic enzymology that directed evolution can provide.     

Site-directed mutagenesis of conserved aromatic residues in rat squalene epoxidase

Squalene epoxidase catalyzes the conversion of squalene to (3S)2,3-oxidosqualene, which is a rate-limiting step of the cholesterol biogenesis. To evaluate the importance of conserved aromatic residues, 15 alanine-substituted mutants were constructed and tested for the enzyme activity. Except F203A, all the mutants significantly lost the enzyme activity, confirming the importance of the residues, either for correct folding of the protein, or for the catalytic machinery of the enzyme. Further, interestingly, F223A mutant no longer accepted (3S)2,3-oxidosqualene as a substrate, while Y473A mutant converted (3S)2,3-oxidosqualene to (3S,22S)2,3:22,23-dioxidosqualene twice more efficiently than wild-type enzyme. It is remarkable that the single amino acid replacement yielded mutants with altered substrate and product specificities. These aromatic residues are likely to be located at the substrate-binding domain of the active-site, and control the stereochemical course of the enzyme reaction.     

A non-active site mutation in human hypoxanthine guanine phosphoribosyltransferase expands substrate specificity

Human hypoxanthine guanine phosphoribosyltransferase (HGPRT) lacks the ability to phosphoribosylate xanthine, a property exhibited by HGPRTs from many parasitic protozoa. Using random mutagenesis we have obtained a mutant, F36L, of human HGPRT that phosphoribosylates xanthine. Examination of the structure indicates that F36 does not make direct contact with the purine, but long-range modulation via loop IV, a segment contacting purine at C2 position, could influence substrate specificity. Expanded substrate specificity to include xanthine probably arises from increased flexibility of loop IV as a consequence of mutation at F36. Mutation of the corresponding residue, L44 in Plasmodium falciparum HGPRT, also results in alteration of K(m) and k(cat) for xanthine, substantiating its role in affecting purine base affinity. Our studies show that mutation of this residue in the core of the protein also affects the stability of both enzymes.     

Directed evolution of a thermostable l-aminoacylase biocatalyst

Enzymes from extreme environments possess highly desirable traits of activity and stability for application under process conditions. One such example is l-aminoacylase (E.C. 3.5.1.14) from Thermococcus litoralis (TliACY), which catalyzes the enantioselective amide hydrolysis of N-protected l-amino acids, useful for resolving racemic mixtures in the preparation of chiral intermediates. Variants of this enzyme with improved activity and altered substrate preference are highly desirable. We have created a structural homology model of the enzyme and applied various two different directed evolution strategies to identify improved variants. Mutants P237S and F251Y were 2.4-fold more active towards N-benzoyl valine relative to the wild type at 65 °C. F251 mutations to basic residues resulted in 4.5-11-fold shifts in the substrate preference towards N-benzoyl phenylalanine relative to N-benzoyl valine. The substrate preference of wild type decreases with increasingly branched and sterically hindered substrates. However, the mutant S100T/M106K disrupted this simple trend by selectively improving the substrate preference for N-benzoyl valine, with a >30-fold shift in the ratio of k_cat values for N-benzoyl valine and N-benzoyl phenylalanine. Mutations that favoured N-benzoyl-phenylalanine appeared at the active site entrance, whereas those improving activity towards N-benzoyl-valine occurred in the hinge region loops linking the dimerization and zinc-binding domains in each monomer. These observations support a previously proposed substrate induced conformational transition between open and closed forms of aminoacylases.     

Structural insight into the dioxygenation of nitroarene compounds: the crystal structure of nitrobenzene dioxygenase

Nitroaromatic compounds are used extensively in many industrial processes and have been released into the environment where they are considered environmental pollutants. 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. However, the bacterium Comamonas sp. strain JS765 can grow with nitrobenzene as a sole source of carbon, nitrogen and energy. Biodegradation is initiated by the nitrobenzene dioxygenase (NBDO) system. We have determined the structure of NBDO, which has a hetero-hexameric structure similar to that of several other Rieske non-heme iron dioxygenases. The catalytic subunit contains a Rieske iron-sulfur center and an active-site mononuclear iron atom. The structures of complexes with substrates nitrobenzene and 3-nitrotoluene reveal the structural basis for its activity with nitroarenes. The substrate pocket contains an asparagine residue that forms a hydrogen bond to the nitro-group of the substrate, and orients the substrate in relation to the active-site mononuclear iron atom, positioning the molecule for oxidation at the nitro-substituted carbon.     

Bacterial metabolism of polycyclic aromatic hydrocarbons: strategies for bioremediation

Polycyclic aromatic hydrocarbons (PAHs) are compounds of intense public concern due to their persistence in the environment and potentially deleterious effects on human, environmental and ecological health. The clean up of such contaminants using invasive technologies has proven to be expensive and more importantly often damaging to the natural resource properties of the soil, sediment or aquifer. Bioremediation, which exploits the metabolic potential of microbes for the clean-up of recalcitrant xenobiotic compounds, has come up as a promising alternative. Several approaches such as improvement in PAH solubilization and entry into the cell, pathway and enzyme engineering and control of enzyme expression etc. are in development but far from complete. Successful application of the microorganisms for the bioremediation of PAH-contaminated sites therefore requires a deeper understanding of the physiology, biochemistry and molecular genetics of potential catabolic pathways. In this review, we briefly summarize important strategies adopted for PAH bioremediation and discuss the potential for their improvement.     

Directed evolution of a ring-cleaving dioxygenase for polychlorinated biphenyl degradation

DoxG, an extradiol dioxygenase involved in the aerobic catabolism of naphthalene, possesses a weak ability to cleave 3,4-dihydroxybiphenyls (3,4-DHB), critical polychlorinated biphenyl metabolites. A directed evolution strategy combining error-prone PCR, saturation mutagenesis, and DNA shuffling was used to improve the polychlorinated biphenyl-degrading potential of DoxG. Screening was facilitated through analysis of filtered, digital imaging of plated colonies. A simple scheme, which is readily adaptable to other activities, enabled the screening of >10(5) colonies/h. The best variant, designated DoxGSMA2, cleaved 3,4-DHB with an apparent specificity constant of 2.0 +/- 0.3 x 10(6) m(-1) s(-1), which is 770 times that of wild-type (WT) DoxG. The specificities of DoxGSMA2 for 1,2-DHN and 2,3-DHB were increased by 6.7-fold and reduced by 2-fold, respectively, compared with the WT enzyme. DoxGSMA2 contained three substituted residues with respect to the WT enzyme: L190M, S191W, and L242S. Structural data indicate that the side chains of residues 190 and 242 occur on opposite walls of the substrate binding pocket and may interact directly with the distal ring of 3,4-DHB or influence contacts between this substrate and other residues. Thus, the introduction of two bulkier residues on one side of the substrate binding pocket and a smaller residue on the other may reshape the binding pocket and alter the catalytically relevant interactions of 3,4-DHB with the enzyme and dioxygen. Kinetic analyses reveal that the substitutions are anti-cooperative.     

Modified substrate specificity of pyrroloquinoline quinone glucose dehydrogenase by biased mutation assembling with optimized amino acid substitution

A biased mutation-assembling method—that is, a directed evolution strategy to facilitate an optimal accumulation of multiple mutations on the basis of additivity principles, was applied to the directed evolution of water-soluble PQQ glucose dehydrogenase (PQQGDH-B) to reduce its maltose oxidation activity, which can lead to errors in blood glucose determination. Mutations appropriate for the reduction without fatal deterioration of its glucose oxidation activity were developed by an error-prone PCR method coupled with a saturation mutagenesis method. Moreover, two types of incorporation frequency based on their contribution were assigned to the mutations: high (80%) and evens (50%), in constructing a multiple mutant library. The best mutant created showed a marked reduction in maltose oxidation activity, corresponding to 4% of that of the wild-type enzyme, with 35% retention of glucose oxidation activity. In addition, this mutant showed a reduction in galactose oxidation activity corresponding to 5% of that of the wild-type enzyme. In conclusion, we succeeded in developing the PQQGDH-B mutants with improved substrate specificity and validated our method coupled with optimized mutations and their contribution-based incorporation frequencies by applying it to the development.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Genetic diversity of dioxygenase genes in polycyclic aromatic hydrocarbon-degrading bacteria isolated from mangrove sediments

To investigate the diversity of dioxygenase genes involved in polycyclic aromatic hydrocarbon (PAH)-degradation, a total of 32 bacterial strains were isolated from surface mangrove sediments, from the genera Mycobacterium, Sphingomonas, Terrabacter, Sphingopyxis, Sphingobium and Rhodococcus. Two sets of PCR primers were constructed to detect the nidA-like and nahAc-like sequences of the alpha subunit of the PAH ring-hydroxylating dioxygenase. PCR amplified the DNA fragments from all Gram-positive bacteria by using nidA-like primers and from all Gram-negative bacteria, except two, by using nahAc-like primers. The nidA-like primers showed three subtypes of nidA-like gene: (i) fadA1, clustering with nidA3 from M. vanbaalenii PYR-1, (ii) nidA, clustering with nidA from PYR-1, and (iii) fadA2 clustering with dioxygenase from Arthrobacter sp. FB24. The amplicons detected by nahAc-like primers had high sequence homologies to phnA1a from Sphingomonas sp. CHY-1 and were amplifiable from 8 of the 16 Gram-negative isolates. The primer also generated amplicons that had a 32-36% similarity to phnA1a and 53-93% identity to p-cumate dioxygenase. These results suggest that the nidA-like and nahAc-like genes are prevalent in the PAH-degrading bacteria and that they are useful for determining the presence of PAH-dioxygenase genes in environmental samples.     

Directed evolution of biphenyl dioxygenase: emergence of enhanced degradation capacity for benzene, toluene, and alkylbenzenes

Biphenyl dioxygenase (Bph Dox) catalyzes the initial oxygenation of biphenyl and related compounds. Bph Dox is a multicomponent enzyme in which a large subunit (encoded by the bphA1 gene) is significantly responsible for substrate specificity. By using the process of DNA shuffling of bphA1 of Pseudomonas pseudoalcaligenes KF707 and Burkholderia cepacia LB400, a number of evolved Bph Dox enzymes were created. Among them, an Escherichia coli clone expressing chimeric Bph Dox exhibited extremely enhanced benzene-, toluene-, and alkylbenzene-degrading abilities. In this evolved BphA1, four amino acids (H255Q, V258I, G268A, and F277Y) were changed from the KF707 enzyme to those of the LB400 enzyme. Subsequent site-directed mutagenesis allowed us to determine the amino acids responsible for the degradation of monocyclic aromatic hydrocarbons.     

Multiple mutations at the active site of naphthalene dioxygenase affect regioselectivity and enantioselectivity

The importance of five amino acids at the active site of the multicomponent naphthalene dioxygenase (NDO) system was determined by generating site-directed mutations in various combinations. The substrate specificities of the mutant enzymes were tested with the substrates indole, indoline, 2-nitrotoluene (2NT), naphthalene, biphenyl, and phenanthrene. Transformation of these substrates measured the ability of the mutant enzymes to catalyze dioxygenation, monooxygenation, and desaturation reactions. In addition, the position of oxidation and the enantiomeric composition of products were characterized. All enzymes with up to three amino acid substitutions were able to catalyze dioxygenation reactions. A subset of these enzymes could also catalyze the monooxygenation of 2NT and desaturation of indoline. Single amino acid substitutions at positions 352 and 206 had the most profound effects on product formation. Of the single mutations made, only changes at position 352 affected the stereochemistry of naphthalene cis-dihydrodiol formed from naphthalene, but in the presence of the F352I mutation, changes at positions 206 and 295 also affected enantioselectivity. Major shifts in regioselectivity with biphenyl and phenanthrene resulted with several of the singly, doubly, and triply mutated enzymes. A new product not formed by the wild-type enzyme, phenanthrene cis-9,10-dihydrodiol, was formed as a major product from phenanthrene by enzymes with two (A206I/F352I) or three amino acid substitutions (A206I/F352I/H295I). The results indicate that a variety of amino acid substitutions are tolerated at the active site of NDO. Journal of Industrial Microbiology & Biotechnology (2001) 27, 94–103. Received 25 September 2000/ Accepted in revised form 29 June 2001     

Directed evolution of LuxI for enhanced OHHL production

Quorum sensing is a common mechanism used by bacteria to coordinate population behavior, and is involved in a variety of biological processes, such as bioluminescence, virulence factor synthesis, antibiotic production, and biofilm formation. To engineer the LuxI enzyme of the LuxI-LuxR quorum-sensing system, we developed a high throughput genetic selection to identify LuxI mutants with improved OHHL (3-oxo-hexanoyl homoserine lactone) synthesis in E. coli. Using this genetic selection, we created LuxI mutants with improved OHHL synthesis rates and yields through directed evolution, identifying three LuxI mutants after two generations. An in vivo semi-quantitative method allowed for verification of the genetic screen and OHHL yields were quantified using HPLC-MS/MS, revealing an 80-fold increase in a mutant culture compared to the wildtype culture. In addition to OHHL, the yields of C6HSL (hexanoyl homoserine lactone) and C8HSL (octanoyl homoserine lactone) were also improved, and a slight change in substrate specificity towards C6HSL production was observed. Based on alignment with the crystal structure of EsaI, a homolog of LuxI, two mutations are most likely involved in enhancing the interactions between the enzyme and the substrates. The high throughput genetic selection and the semi-quantitative method can be conveniently modified for the directed evolution of LuxI homologs. The identification of these LuxI mutants has implications in synthetic biology, where they can be used for the construction of artificial genetic circuits. In addition, development of drugs that specifically target quorum sensing to attenuate the pathogenesis of gram-negative infectious bacteria might also benefit from the insights into the molecular mechanism of quorum sensing revealed by the amino acid substitutions.     

Biochemical characterization of L-DOPA 2,3-dioxygenase, a single-domain type I extradiol dioxygenase from lincomycin biosynthesis

l-DOPA-2,3-dioxygenase from Streptomyces lincolnensis is a single-domain type I extradiol dioxygenase of the vicinal oxygen chelate superfamily and catalyzes the second step in the metabolism of tyrosine to the propylhygric acid moiety of the antibiotic, lincomycin. S. lincolnensisl-DOPA-2,3-dioxygenase was overexpressed, purified and reconstituted with Fe(II). The activity of l-DOPA-2,3-dioxygenase was kinetically characterized with l-DOPA (K_M = 38 μM, k_cat = 4.2 min⁻¹) and additional catecholic substrates including dopamine, 3,4-dihydroxyhydrocinnamic acid, catechol and d-DOPA. 3,4-Dihydroxyphenylacetic acid was characterized as a competitive inhibitor of the enzyme (K_i = 2.2 mM). Site-directed mutagenesis and its effects on enzymatic activity were used to identify His14 and His70 as iron ligands.     

Metabolic engineering of Arabidopsis for remediation of different polycyclic aromatic hydrocarbons using a hybrid bacterial dioxygenase complex

The widespread presence of polycyclic aromatic hydrocarbons (PAHs) and their potential harm to various organisms has generated interest in efficiently eliminating these compounds from the environment. Phytoremediation is an efficient technology for cleaning up pollutants. However, unlike microorganisms, plants lack the catabolic pathway for complete degradation of these dangerous groups of compounds. One way to enhance the potential of plants for remediation of these compounds is by transferring genes involved in xenobiotic degradation from microbes to plants. In this paper, four genes, namely nidA and nidB (encoding the large and small subunits of naphthalene dioxygenase of Mycobacterium vanbaalenii PYR-1) as well as NahAa and NahAb (encoding flavoprotein reductase and ferredoxin of the electron-transport chain of the Pseudomonas putida G7 naphthalene dioxygenase system), were transferred and ectopically expressed in Arabidopsis thaliana. Transgenic Arabidopsis plants overexpressing the heterozygous naphthalene dioxygenase system exhibited enhanced tolerance toward 2–4 rings PAHs. Transgenic plants assimilated PAHs from the culture media faster and accumulated less in vivo than wild-type plants. Furthermore, examination of metabolic intermediates by gas chromatography–mass spectrometry revealed that the naphthalene metabolic pathway in transgenic plants mainly involves the dioxygenase pathway. Taken together, our findings suggest that grafting the naphthalene dioxygenase complex into plants is a possible strategy to breed PAH-tolerant plants to efficiently degrade PAHs in the environment.