Toluene Dioxygenase Research Articles

Microbial Arene Oxidations

AbstractFor Abstract see ChemInform Abstract in Full Text.

Enzymatic oxidation of thioanisoles: isolation and absolute configuration of metabolites

Oxidation of p-bromothioanisole with toluene dioxygenase provides the corresponding diene diol 2 in good yield. Electrochemical reduction of 2 gives access to diene diol 3, which is not accessible by direct bio-oxidation of thioanisole. Absolute configuration and enantiomeric purity are reported for the new metabolites.

Biocatalytic synthesis of polycatechols from toxic aromatic compounds.

A process is described in which toxic aromatic compounds are converted by toluene dioxygenase and in turn toluene cis-dihydrodiol dehydrogenase to catechols which are further polymerized by peroxidase-catalyzed oxidation producing polycatechols. Three approaches for obtaining catechols were employed: (1) addition of halogenated aromatics to P. putida F1, resulting in the accumulation of halogenated catechols; (2) inhibition of catechol 2,3-dioxygenase of P. putida F1 by known aromatic and aliphatic inhibitors; and (3) overexpression of toluene dioxygenase and toluene cis-dihydrodiol dehydrogenase genes in E. coli JM109. The process is suitable for producing novel catechols that upon oxidation may yield polymers with unique properties, presenting a tool for producing tailor-made biopolymers. Formation of 3-chlorocatechol from chlorobenzene, 3,4-dichlorocatechol from 1,2-dichlorobenzene, and catechol from benzene and their subsequent oxidation and polymerization was demonstrated. Oxidation of catechol yielded polymers with molecular weights of up to 4000 Daltons. Their apparently high water solubility eliminates the need for water-miscible solvents. In aqueous solution oxidation of catechols was rapid, yet the presence of 20%, 30%, and 40% ethanol, resulted in a rate decrease of 31%, 95%, and 93%, respectively. The advantage is that significantly less peroxidase is required for performing the reactions if miscible solvents are not employed. Furthermore, water-soluble polymers may be desirable for many applications.

Concise chemoenzymatic synthesis of epi-inositol

epi-Inositol was synthesized in six steps in 40% overall yield from a bacterial bromobenzene metabolite. The chemoenzymatic route involved toluene dioxygenase oxidation, substrate-directed catalytic osmylation, m-CPBA epoxidation, radical debromination, and Amberlite-catalized hydrolysis. The route described is amenable to scaleup and could allow access to cis-inositol, and deoxy derivatives of epi-inositol.

Effect of ethanol, acetate, and phenol on toluene degradation activity and tod-lux expression in Pseudomonas putida TOD102: evaluation of the metabolic flux dilution model.

The reporter strain Pseudomonas putida TOD102 (with a tod-lux fusion) was used in chemostat experiments with binary substrate mixtures to investigate the effect of potentially occurring cosubstrates on toluene degradation activity. Although toluene was simultaneously utilized with other cosubstrates, its metabolic flux (defined as the toluene utilization rate per cell) decreased with increasing influent concentrations of ethanol, acetate, or phenol. Three inhibitory mechanisms were considered to explain these trends: (1) repression of the tod gene (coding for toluene dioxygenase) by acetate and ethanol, which was quantified by a decrease in specific bioluminescence; (2) competitive inhibition of toluene dioxygenase by phenol; and (3) metabolic flux dilution (MFD) by all three cosubstrates. Based on experimental observations, MFD was modeled without any fitting parameters by assuming that the metabolic flux of a substrate in a mixture is proportional to its relative availability (expressed as a fraction of the influent total organic carbon). Thus, increasing concentrations of alternative carbon sources "dilute" the metabolic flux of toluene without necessarily repressing tod, as observed with phenol (a known tod inducer). For all cosubstrates, the MFD model slightly overpredicted the measured toluene metabolic flux. Incorporating catabolite repression (for experiments with acetate or ethanol) or competitive inhibition (for experiments with phenol) with independently obtained parameters resulted in more accurate fits of the observed decrease in toluene metabolic flux with increasing cosubstrate concentration. These results imply that alternative carbon sources (including inducers) are likely to hinder toluene utilization per unit cell, and that these effects can be accurately predicted with simple mathematical models.

Addition of Aromatic Substrates Restores Trichloroethylene Degradation Activity in Pseudomonas putida F1

The rate of trichloroethylene (TCE) degradation by toluene dioxygenase (TDO) in resting cells of Pseudomonas putida F1 gradually decreased and eventually stopped within 1.5 h, as in previous reports. However, the subsequent addition of toluene, which is the principal substrate of TDO, resulted in its immediate degradation without a lag phase. After the consumption of toluene, degradation of TCE restarted at a rate similar to its initial degradation, suggesting that this degradation was mediated by TDO molecules that were present before the cessation of TCE degradation. The addition of benzene and cumene, which are also substrates of TDO, also caused restoration of TCE degradation activity: TCE was degraded simultaneously with cumene, and a larger amount of TCE was degraded after cumene was added than after toluene or benzene was added. But substrates that were expected to supply the cells with NADH or energy did not restore TCE degradation activity. This cycle of pseudoinactivation and restoration of TCE degradation was observed repeatedly without a significant decrease in the number of viable cells, even after six additions of toluene spread over 30 h. The results obtained in this study demonstrate a new type of restoration of TCE degradation that has not been previously reported.

A survey of indigenous microbial hydrocarbon degradation genes in soils from Antarctica and Brazil.

Total community DNA from 29 noncontaminated soils and soils impacted by petroleum hydrocarbons and chloro-organics from Antarctica and Brazil were screened for the presence of nine catabolic genes, encoding alkane monooxygenase or aromatic dioxygenases, from known bacterial biodegradation pathways. Specific primers and probes targeting alkane monooxygenase genes were derived from Pseudomonas putida ATCC 29347 (Pp alkB), Rhodococcus sp. strain Q15 (Rh alkB1, Rh alkB2), and Acinetobacter sp. ADP-1 (Ac alkM). In addition, primers and probes detecting aromatic dioxygenase genes were derived from P. putida ATCC 17484 (ndoB), P. putida F1 (todC1), P. putida ATCC 33015 (xylE and cat23), and P. pseudoalcaligenes KF707 (bphA). The primers and probes were used to analyze total community DNA extracts by using PCR and hybridization analysis. All the catabolic genes, except the Ac alkM, were detected in contaminated and control soils from both geographic regions, with a higher frequency in the Antarctic soils. The alkane monooxygenase genes, Rh alkB1 and Rh alkB2, were the most frequently detected alk genes in both regions, while Pp alkB was not detected in Brazil soils. Genes encoding the aromatic dioxygenases toluene dioxygenase (todC1) and biphenyl dioxygenase (bphA) were the most frequently detected in Antarctica, and todC1 and catechol-2,3-dioxygenase (cat23) were the most frequent in Brazil soils. Hybridization analysis confirmed the PCR results, indicating that the probes used had a high degree of homology to the genes detected in the soil extracts and were effective in detecting biodegradative potential in the indigenous microbial population.

Aerobic and anaerobic toluene degradation by a newly isolated denitrifying bacterium, Thauera sp. strain DNT-1.

A newly isolated denitrifying bacterium, Thauera sp. strain DNT-1, grew on toluene as the sole carbon and energy source under both aerobic and anaerobic conditions. When this strain was cultivated under oxygen-limiting conditions with nitrate, first toluene was degraded as oxygen was consumed, while later toluene was degraded as nitrate was reduced. Biochemical observations indicated that initial degradation of toluene occurred through a dioxygenase-mediated pathway and the benzylsuccinate pathway under aerobic and denitrifying conditions, respectively. Homologous genes for toluene dioxygenase (tod) and benzylsuccinate synthase (bss), which are the key enzymes in aerobic and anaerobic toluene degradation, respectively, were cloned from genomic DNA of strain DNT-1. The results of Northern blot analyses and real-time quantitative reverse transcriptase PCR suggested that transcription of both sets of genes was induced by toluene. In addition, the tod genes were induced under aerobic conditions, whereas the bss genes were induced under both aerobic and anaerobic conditions. On the basis of these results, it is concluded that strain DNT-1 modulates the expression of two different initial pathways of toluene degradation according to the availability of oxygen in the environment.

Dioxygenase-catalysed oxidation of alkylaryl sulfides: sulfoxidation versus cis-dihydrodiol formation.

Toluene- and naphthalene-dioxygenase-catalysed sulfoxidation of nine disubstituted methylphenyl sulfides, using whole cells of Pseudomonas putida, consistently gave the corresponding enantioenriched sulfoxides. Using the P. putida UV4 mutant strain, and these substrates, differing proportions of the corresponding cis-dihydrodiol sulfides were also isolated. Evidence was found for the concomitant dioxygenase-catalysed cis-dihydroxylation and sulfoxidation of methyl para-tolyl sulfide. A simultaneous stereoselective reductase-catalysed deoxygenation of (S)-methyl para-tolyl sulfoxide, led to an increase in the proportion of the corresponding cis-dihydrodiol sulfide. The enantiopurity values and absolute configurations of the corresponding cis-dihydrodiol metabolites from methyl ortho- and para-substituted phenyl sulfides were determined by different methods, including chemoenzymatic syntheses from the cis-dihydrodiol metabolites of para-substituted iodobenzenes. Further evidence was provided to support the validity of an empirical model to predict, (i) the stereochemistry of cis-dihydroxylation of para-substituted benzene substrates, and (ii) the regiochemistry of cis-dihydroxylation reactions of ortho-substituted benzenes, each using toluene dioxygenase as biocatalyst.

Total degradation of pentachloroethane by an engineered Alcaligenes strain expressing a modified camphor monooxygenase and a hybrid dioxygenase.

We engineered biphenyl-degrading Alcaligenes sp. strain KF711 for total degradation of pentachloroethane (PCA), which expresses a modified camphor monooxygenase and a hybrid dioxygenase consisting of TodC1 (a large subunit of toluene dioxygenase of Pseudomonas putida F1) and BphA2-BphA3-pbhA4 (a small subunit, ferredoxin and ferredoxin reductase of biphenyl dioxygenase, respectively, in strain KF707). Modified camphor monooxygenase genes (camCAB) were supplied as a plasmid and the todC1 gene was integrated within the chromosomal bph gene cluster by a single crossover recombination. The resultant strain KF711S-3cam dechlorinated PCA to trichloroethene by the action of the modified camphor monooxygenase under anaerobic conditions. The same strain subsequently degraded trichloroethene formed oxidatively by the action of the Tol-Bph hybrid dioxygenase under aerobic conditions. Thus sequential anaerobic and aerobic treatments of the KF711S-3cam resting cells resulted in efficient and total degradation of PCA.

Directed evolution of the dioxygenase complex for the synthesis of furanone flavor compounds

Herein we report a new preparation of 4-hydroxy-2,5-dimethyl-2,3-dihydrofuran-3-one, the flavor compound strawberry furanone, based on a ‘green’ approach with a minimum number of steps. The first step is an enzymatic dioxygenation of p-xylene to form cyclohexadiene- cis-diol, followed by ring opening via ozonolysis, and ring closure to form the furanone. In efforts to improve the efficiency of the enzymatic step, a directed evolution approach was taken to increase the substrate specificity and selectivity of the toluene dioxygenase enzyme system.

Genes similar to naphthalene dioxygenase genes in trifluralin-degrading bacteria.

Trifluralin (alpha,alpha,alpha-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine) is a dinitroaniline compound which was first produced in the 1960s and has been used extensively as an agricultural herbicide. There are a few publications on the biodegradation of this xenobiotic compound, but to our knowledge nothing has been documented on the genetic aspects of its catabolism. In this article, we report the analysis of DNA isolated from bacteria previously shown to degrade trifluralin, using as probes the catabolic genes ndoB, todC, xyIX, catA and xyIE which encode the enzymes naphthalene 1,2-dioxygenase, toluene dioxygenase, toluate 1,2-dioxygenase, catechol 1,2-dioxygenase and catechol 2,3-dioxygenase respectively. Using PCR and hybridization analysis, the strong hybridization of the ndoB gene with DNA extracted from four trifluralin-degrading isolates was demonstrated, although none of them was able to degrade naphthalene, as indicated by the 'clear zone' test. The results indicated the presence in these bacteria of a dioxygenase gene, whose product could act on trifluralin as its principal substrate, or fortuitously, by cometabolism. This is the first publication on genes in trifluralin-degrading bacteria.

Using a residue clash map to functionally characterize protein recombination hybrids

In this article, we introduce a rapid, protein sequence database-driven approach to characterize all contacting residue pairs present in protein hybrids for inconsistency with protein family structural features. This approach is based on examining contacting residue pairs with different parental origins for different types of potentially unfavorable interactions (i.e. electrostatic repulsion, steric hindrance, cavity formation and hydrogen bond disruption). The identified clashing residue pairs between members of a protein family are then contrasted against functionally characterized hybrid libraries. Comparisons for five different protein recombination studies available in the literature: (i) glycinamide ribonucleotide transformylase (GART) from Escherichia coli (purN) and human (hGART), (ii) human Mu class glutathione S-transferase (GST) M1-1 and M2-2, (iii) beta-lactamase TEM-1 and PSE-4, (iv) catechol-2,3-oxygenase xylE and nahH, and (v) dioxygenases (toluene dioxygenase, tetrachlorobenzene dioxygenase and biphenyl dioxygenase) reveal that the patterns of identified clashing residue pairs are remarkably consistent with experimentally found patterns of functional crossover profiles. Specifically, we show that the proposed residue clash maps are on average 5.0 times more effective than randomly generated clashes and 1.6 times more effective than residue contact maps at explaining the observed crossover distributions among functional members of hybrid libraries. This suggests that residue clash maps can provide quantitative guidelines for the placement of crossovers in the design of protein recombination experiments.

Intrinsic bioremediability of an aromatic hydrocarbon-polluted groundwater: diversity of bacterial population and toluene monoxygenase genes.

The functional and phylogenetic biodiversity of bacterial communities in a benzene, toluene, ethylbenzene and xylene (BTEX)-polluted groundwater was analysed. To evaluate the feasibility of using an air sparging treatment to enhance bacterial degradative capabilities, the presence of degrading microorganisms was monitored. The amplification of gene fragments corresponding to toluene monooxygenase (tmo), catechol 1,2-dioxygenase, catechol 2,3-dioxygenase and toluene dioxygenase genes in DNA extracted directly from the groundwater samples was associated with the presence of indigenous degrading bacteria. Five months of air injection reduced species diversity in the cultivable community (as calculated by the Shannon-Weaver index), while little change was noted in the degree of biodiversity in the total bacterial community, as characterised by denaturing gradient gel electrophoresis (DGGE) analysis. BTEX-degrading strains belonged to the genera Pseudomonas, Microbacterium, Azoarcus, Mycobacterium and Bradyrhizobium. The degrading capacities of three strains in batch liquid cultures were also studied. In some of these microorganisms different pathways for toluene degradation seemed to operate simultaneously. Pseudomonas strains of the P24 operational taxonomic unit, able to grow only on catechol and not on BTEX, were the most abundant, and were present in the groundwater community at all stages of treatment, as evidenced both by cultivation approaches and by DGGE profiles. The presence of different tmo-like genes in phylogenetically distant strains of Pseudomonas, Mycobacterium and Bradyrhizobium suggested recent horizontal gene transfer in the groundwater.

Detection of dioxygenase genes present in various activated sludge.

Activated sludge from refineries contains various microorganisms that could utilize aromatics under aerobic conditions due to the oxygenase enzymes. Dioxygenase enzymes are oxygenases, which are involved in the ring cleavage step of aromatic hydrocarbons. In this study, the selected catabolic loci involved in ring cleavage have been monitored in the activated sludge samples at different time intervals. The investigation of the dioxygenase genes in the Effluent Treatment Plants (ETPs) and evaluation of their presence at different time points provides a clue for the aromatic utilizing potential of the inherent microbial flora. The catabolic gene loci pheB, xylE, tod-isp, bed and nahG responsible for the enzymes catechol 1,2-dioxygenase, catechol 2,3-dioxygenase, toluene dioxygenase-iron-sulphur protein component, benzene dioxygenase and naphthalene dioxygenase were used respectively. The time dependent change in eubacterial population was demonstrated by the amplification of 16S rDNA product, followed by restriction digestion. The template DNA was obtained from the activated sludge collected from ETPs. The supporting physiological data for the overall performance of sludge was developed using respirometric analysis. The on-site COD and MLSS analysis for ETP was used in final evaluation. The study was carried out with samples collected from three different ETPs and also from a selected ETP at different time intervals. The respirometric studies were carried out with phenol, catechol, toluene, and naphthalene to arrive at the target genotypes for further study by PCR protocol. The respirometric analysis coupled with the COD and MLSS analysis represented the physiological capacity of the various sludges. Initially, the tracking protocol was optimized by using different sludge samples, which were collected from refineries. The selected genotypes were amplified and their presence has been confirmed using Southern analysis. The gene loci tod-isp, bed and xylE were commonly observed at various time intervals of the sludge from the same source. The gene loci pheB and nahG were found to be relatively rare. The 16S rDNA PCR products after restriction digestion produced different DNA fingerprint patterns, suggesting that the microbial community composition was diverse in the three sources. Similarly, the presence of the catechol 2,3-dioxygenase, benzene dioxygenase and toluene dioxygenase genes confirmed the aromatic degrading potential in the various sludges. The probes could not pick the nahG and pheB genes. However, the respirometeric assay suggested that the oxidative capacity to use naphthalene as a substrate exists. Our study of the diversity at various time points from the ETP provided an overview of the shifts of the catabolic composition of the sludge. This also depends on the influential parameters like the incoming pollutant level and the environmental conditions that are prevailing and often changing from time to time. The results of direct DNA extraction and PCR amplification do reflect the relative abundance of a particular catabolic genotype, which could be used to monitor the efficiency of treatment.

Genetic engineering of a highly solvent-tolerant Pseudomonas putida strain for biotransformation of toluene to p-hydroxybenzoate.

The solvent-tolerant strain Pseudomonas putida DOT-T1E has been engineered for biotransformation of toluene into 4-hydroxybenzoate (4-HBA). P. putida DOT-T1E transforms toluene into 3-methylcatechol in a reaction catalyzed by toluene dioxygenase. The todC1C2 genes encode the alpha and beta subunits of the multicomponent enzyme toluene dioxygenase, which catalyzes the first step in the Tod pathway of toluene catabolism. A DOT-T1EdeltatodC mutant strain was constructed by homologous recombination and was shown to be unable to use toluene as a sole carbon source. The P. putida pobA gene, whose product is responsible for the hydroxylation of 4-HBA into 3,4-hydroxybenzoate, was cloned by complementation of a Pseudomonas mendocina pobA1 pobA2 double mutant. This pobA gene was knocked out in vitro and used to generate a double mutant, DOT-T1EdeltatodCpobA, that was unable to use either toluene or 4-HBA as a carbon source. The tmo and pcu genes from P. mendocina KR1, which catalyze the transformation of toluene into 4-HBA through a combination of the toluene 4-monoxygenase pathway and oxidation of p-cresol into the hydroxylated carboxylic acid, were subcloned in mini-Tn5Tc and stably recruited in the chromosome of DOT-T1EdeltatodCpobA. Expression of the tmo and pcu genes took place in a DOT-T1E background due to cross-activation of the tmo promoter by the two-component signal transduction system TodST. Several independent isolates that accumulated 4-HBA in the supernatant from toluene were analyzed. Differences were observed in these clones in the time required for detection of 4-HBA and in the amount of this compound accumulated in the supernatant. The fastest and most noticeable accumulation of 4-HBA (12 mM) was found with a clone designated DOT-T1E-24.

Production of dyestuffs from indole derivatives by naphthalene dioxygenase and toluene dioxygenase.

To isolate and characterize the phorate [O,O-diethyl-S-(ethylthio)methyl phosphoradiothioate] degrading bacteria from agricultural soil, and their assessment for multifarious biological activities of environmental and agronomic significance. Based on their morphological and biochemical characteristics, the selected isolates PS-1, PS-2 and PS-3 were presumptively identified as Rhizobium, Pseudomonas and Proteus species, respectively. The HPLC analysis of phorate in bioaugmented soil revealed its complete disappearance within 40 days. The degradation isotherms of the isolates PS-1, PS-2 and PS-3 suggested time-dependent disappearance of phorate following the first-order rate kinetics at the corresponding rate constants of 0.04, 0.05 and 0.04 d-1. Besides, the isolates concurrently exhibited substantial phosphate solubilization, indole acetic acid (IAA) and siderophore production, as well as limited biocontrol activity against fungal phytopathogens. The data revealed the potential of isolates for collateral plant growth promotion, biocontrol and bioremediation. The selected strains may serve as an important bioresource for development of effective super-bioinoculants.

Stereochemical and mechanistic aspects of dioxygenase-catalysed benzylic hydroxylation of indene and chromane substrates.

Toluene dioxygenase (TDO)-catalysed benzylic hydroxylation of indene substrates (8, 16 and 17), using whole cell cultures of Pseudomonas putida UV4, was found to yield inden-1-ol (14 and 22) and indan-1-one bioproducts (15 and 23). The formation of these bioproducts is consistent with the involvement of carbon-centred radical intermediates. TDO-catalysed oxidation of indenes 8 and 16 also gave cis-diols 13 and 18 respectively. TDO and naphthalene dioxygenase (NDO), used as both whole-cell preparations and as purified enzymes, were found to catalyse the benzylic hydroxylation of chromane 30, deuteriated (+/-)-chromane 30D and enantiomers (4S)-30D and (4R)-30D to yield (4R)- and (4S)-chroman-4-ols 31/31D respectively. The mechanism of benzylic hydroxylation of chromane 30/30D involves the stereoselective abstraction of a pro-R (with TDO) or a pro-S (with NDO) hydrogen atom at C-4 and a marked preference for retention of configuration.

Enantiocomplementary Preparation of (S)‐ and (R)‐1‐Pyridylalkanols via Ketone Reduction and Alkane Hydroxylation Using Whole Cells of Pseudomonas putida UV4.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Dioxygenase-catalysed oxidation of monosubstituted thiophenes: sulfoxidation versus dihydrodiol formation.

Toluene dioxygenase (TDO)-catalysed sulfoxidation, using Pseudomonas putida UV4, was observed for the thiophene substrates 1A-1N. The unstable thiophene oxide metabolites, 6A-6G, 6K-6N, spontaneously dimerised yielding the corresponding racemic disulfoxide cycloadducts 7A-7G, 7K-7N. Dimeric or crossed [4 + 2] cycloaddition products, derived from the thiophene oxide intermediates 6A and 6D or 6B and 6D, were found when mixtures of thiophene substrates 1A and 1D or 1B and 1D were biotransformed. The thiophene sulfoxide metabolite 6B was also trapped as cycloadducts 17 or 18 using stable dienophiles. Preferential dioxygenase-catalysed oxidation of the substituent on the thiophene ring, including exocyclic sulfoxidation (1H-1J) and cis-dihydroxylation of a phenyl substituent (1G and 1N), was also observed. An enzyme-catalysed deoxygenation of a sulfoxide in P. putida UV4 was noticed when racemic disulfoxide cyclo-adducts 7A, 7B and 7K were converted to the corresponding enantioenriched monosulfoxides 8A, 8B and 8K via a kinetic resolution process. The parent thiophene 1A and the 3-substituted thiophenes 1K-1N were also found to undergo ring dihydroxylation yielding the cis/trans-dihydrodiol metabolites 9A and 9K-9N. Evidence is provided for a dehydrogenase-catalysed desaturation of a heterocyclic dihydrodiol (9Kcis/9Ktrans) to yield the corresponding 2,3-dihydroxythiophene (24) as its preferred thiolactone tautomer (23). A simple model to allow prediction of the structure of metabolites, formed from TDO-catalysed bacterial oxidation of thiophene substrates 1, is presented.