Oxidation of aliphatic olefins by toluene dioxygenase: enzyme rates and product identification

Lange, Cleston C.; Wackett, Lawrence P.

doi:10.1128/jb.179.12.3858-3865.1997

Cited by 41 publications

(29 citation statements)

References 27 publications

(17 reference statements)

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“…With an initial rate of product formation of 0.22 mmol of 3-hydroxy-4-picoline/h/g of dried cells, TDO is ϳ4.5% as active on the heterocyclic substrate as it is towards toluene (5.1 mmol of toluene cis-dihydrodiol/h/g of dried cells). The polar nitrogen-containing heterocycle is not transformed as efficiently as are other nonnatural TDO substrates, such as aromatics with bulky substituents and aliphatic olefins (12,22,33) Directed evolution of TDO. Oxygenase activity is often determined by monitoring consumption of exogenously added NADH (16).…”

Section: Resultsmentioning

confidence: 99%

Laboratory Evolution of Toluene Dioxygenase To Accept 4-Picoline as a Substrate

Sakamoto

Joern

Arisawa

et al. 2001

Appl Environ Microbiol

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We are using directed evolution to extend the range of dioxygenase-catalyzed biotransformations to include substrates that are either poorly accepted or not accepted at all by the naturally occurring enzymes. Here we report on the oxidation of a heterocyclic substrate, 4-picoline, by toluene dioxygenase (TDO) and improvement of the enzyme's activity by laboratory evolution. The biotransformation of 4-picoline proceeds at only ϳ4.5% of the rate of the natural reaction on toluene. Random mutagenesis, saturation mutagenesis, and screening directly for product formation using a modified Gibbs assay generated mutant TDO 3-B38, in which the wild-type stop codon was replaced with a codon encoding threonine. Escherichia coli-expressed TDO 3-B38 exhibited 5.6 times higher activity toward 4-picoline and ϳ20% more activity towards toluene than wild-type TDO. The product of the biotransformation of 4-picoline is 3-hydroxy-4-picoline; no cis-diols of 4-picoline were observed.Dioxygenase enzymes involved in the catabolism of aromatic hydrocarbons by soil microorganisms nicely illustrate nature's ability to adapt to different carbon sources through evolution of the substrate specificity of the biodegradation enzymes (35). The biodegradation pathway often begins with the dioxygenasecatalyzed regio-and enantio-specific introduction of molecular oxygen into aromatic compounds to form the corresponding arene cis-diols, with consumption of NADH (13, 36) ( Fig. 1).It has been noted that the chiral products of the dioxygenase reaction can be converted in a variety of synthetic reactions to advanced intermediates for natural-product synthesis (8, 15). Toluene dioxygenase (TDO) from Pseudomonas putida readily dihydroxylates aromatic carbocycles with one or two small hydrophobic substituents (6, 33). Activity towards much larger, fused-ring substrates or substrates with polar or bulky substituents is considerably reduced or nonexistent (33).We are attempting to extend the utility of dioxygenase-catalyzed biotransformations by engineering the catalysts to accept a wider range of substrates, including small heterocyclic compounds and polar-substituted carbocyclic aromatics. N-Heterocyclic compounds are useful for the synthesis of biologically active compounds; recent reports describe the regioand/or stereo-controlled biooxidation of N-heterocycles (20,26). Bicyclic compounds such as quinolines and benzofurans are accepted by TDO, with oxygen insertion occurring primarily in the carbocylic rings (3-5).To date, reactions on single-ring heterocycles have not been reported. We have found that TDO catalyzes the oxygenation of 4-picoline (4-methylpyridine), although at a much slower rate than on its preferred substrate toluene.A strategy of DNA shuffling to create libraries of hybrid genes and screening has been used with some success to extend the substrate range of dioxygenases that degrade environmental pollutants such as polychlorinated biphenyls (7, 21). An alternative approach is to fine tune a single gene by accumulating beneficial mutat...

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Section: Resultsmentioning

confidence: 99%

Laboratory Evolution of Toluene Dioxygenase To Accept 4-Picoline as a Substrate

Sakamoto

Joern

Arisawa

et al. 2001

Appl Environ Microbiol

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“…The cell-free crude extract was filtered through a 0.2-m membrane and stored at Ϫ20°C until needed. The partial purification of crude protein extract (0.95 mg ml Ϫ1 ) was carried out by ammonium sulfate precipitation according to the method of Lange and Wackett (15). The protein pellet was resuspended in 100 mM Tris-HCl buffer (pH 7.5) containing 10% ethanol and 10% glycerol (TEG buffer), supplemented with 1 mM dithiothreitol (DTT), at a ratio of 1 g pellet per 2 ml buffer.…”

Section: Methodsmentioning

confidence: 99%

“…However, TCE cometabolic degradation is considered an unsustainable process due to cytotoxicity, inhibition, or inactivation of TCE-degrading enzymes. These phenomena have been observed in studies using both whole cells and purified enzymes, including soluble methane monooxygenases from Methylosinus trichosporium OB3b (9) and Nitrosomonas europaea (13), toluene 2-monooxygenase from Burkholderia cepacia G4 (19,27), toluene dioxygenase (TDO) from Pseudomonas putida F1 (15,18), and butaneoxidizing bacteria, i.e., Pseudomonas butanovora, Mycobacterium vaccae, and Nocardioides sp. CF8 (11).…”

mentioning

confidence: 99%

Cometabolic Degradation of Trichloroethene by Rhodococcus sp. Strain L4 Immobilized on Plant Materials Rich in Essential Oils

Suttinun

Müller

Luepromchai

2010

Appl Environ Microbiol

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The cometabolic degradation of trichloroethene (TCE) by Rhodococcus sp. L4 was limited by the loss of enzyme activity during TCE transformation. This problem was overcome by repeated addition of inducing substrates, such as cumene, limonene, or cumin aldehyde, to the cells. Alternatively, Rhodococcus sp. L4 was immobilized on plant materials which contain those inducers in their essential oils. Cumin seeds were the most suitable immobilizing material, and the immobilized cells tolerated up to 68 M TCE and degraded TCE continuously. The activity of immobilized cells, which had been inactivated partially during TCE degradation, could be reactivated by incubation in mineral salts medium without TCE. These findings demonstrate that immobilization of Rhodococcus sp. L4 on plant materials rich in essential oils is a promising method for efficient cometabolic degradation of TCE.Various bacteria have been reported to degrade trichloroethene (TCE) aerobically via cometabolic degradation with broad-substrate-specificity enzymes (2). However, TCE cometabolic degradation is considered an unsustainable process due to cytotoxicity, inhibition, or inactivation of TCE-degrading enzymes. These phenomena have been observed in studies using both whole cells and purified enzymes, including soluble methane monooxygenases from Methylosinus trichosporium OB3b (9) and Nitrosomonas europaea (13), toluene 2-monooxygenase from Burkholderia cepacia G4 (19, 27), toluene dioxygenase (TDO) from Pseudomonas putida F1 (15, 18), and butaneoxidizing bacteria, i.e., Pseudomonas butanovora, Mycobacterium vaccae, and Nocardioides sp. CF8 (11). Nevertheless, the addition of an inducer or growth substrate can maintain TCE cometabolic degradation. For example, the TCE-degrading activity of P. putida F1 toluene dioxygenase was restored after adding benzene, cumene, or toluene to displace TCE and its reactive intermediates from the enzyme active site (18). Arp et al. (2) suggested that the rate of enzyme maintenance and recovery depended on the extent of inactivation and the balance of TCE and inducer/growth substrate concentrations.Plant essential oils and their components, such as citral, limonene, cumene, and cumin aldehyde, have been found to induce TCE degradation in Rhodococcus sp. L4 (24). However, the removal of TCE by this bacterium was effective only for a short period. The impacts of TCE on Rhodococcus spp. and their enzymes have not been studied in detail, even though many bacteria of this genus exhibited high TCE-degrading activities (i.e., Rhodococcus erythropolis JE 77, R. erythropolis BD2, Rhodococcus sp. Sm-1, and Rhodococcus sp. Wrink) (5,6,7,16). This study therefore investigated the changes in TCE-degrading activity of Rhodococcus sp. L4 cells and TDO during exposure to TCE. Two enzyme maintenance approaches were evaluated, namely, repeated addition of essential oil components to the system and immobilization of the bacterial cells on plant material rich in essential oils. Immobilized microorganisms are generally capable of degrading ...

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“…TOD also acts as a monooxygenase on monocyclic aromatics, aliphatic olefi ns and other miscellaneous substrates [33,34]. By these means it converts different isomers of dimethylbenzene into dimethyl phenols and isomers of nitrotoluene into nitobenzyl alcohols and nitophenols [31,34]. Allylic methyl group monooxygenation can be seen with different halo-propene and halo-butene isomers which are converted into butene-1-ol and propene-1-ol, respectively [35].…”

Section: Dioxygenases: Tod (Ec 11412)mentioning

confidence: 99%

“…TOD acts as a dioxygenase against a range of compounds including monocyclic aromatics, fused aromatics, linked aromatics and aliphatic olefi ns [31,32]. TOD also acts as a monooxygenase on monocyclic aromatics, aliphatic olefi ns and other miscellaneous substrates [33,34]. By these means it converts different isomers of dimethylbenzene into dimethyl phenols and isomers of nitrotoluene into nitobenzyl alcohols and nitophenols [31,34].…”

Section: Dioxygenases: Tod (Ec 11412)mentioning

confidence: 99%