Initial reactions in the oxidation of ethylbenzene by Pseudomonas putida

Gibson, David T.; Gschwendt, Brigitte; Yeh, W K; Kobal, Val M.

doi:10.1021/bi00732a008

Cited by 133 publications

(63 citation statements)

References 20 publications

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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 organism was grown in mineral salts medium (34) or on mineral salts medium agar (2.0%) plates. Toluene was supplied in the vapor phase as previously described (10). Large quantities of cells used in enzyme purification studies were grown with forced aeration at 25°C in 12-liter cultures in a New Brunswick Microferm fermentor.…”

mentioning

confidence: 99%

Toluene-4-monooxygenase, a three-component enzyme system that catalyzes the oxidation of toluene to p-cresol in Pseudomonas mendocina KR1

1991

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Pseudomonas mendocina KR1 grows on toluene as a sole carbon and energy source. A multicomponent oxygenase was partially purified from toluene-grown cells and separated into three protein components. The reconstituted enzyme system, in the presence of NADH and Fe2 , oxidized toluene to p-cresol as the first detectable product. Experiments with p-deuterotoluene led to the isolation of p-cresol which retained 68% of the deuterium initially present in the parent molecule. When the reconstituted enzyme system was incubated with toluene in the presence of 1802, the oxygen in p-cresol was shown to be derived from molecular oxygen. The results demonstrate that P. mendocina KR1 initiates degradation of toluene by a multicomponent enzyme system which has been designated toluene-4-monooxygenase.Several small aromatic hydrocarbons can be utilized by bacteria as sole carbon and energy sources for growth. Degradation of these hydrocarbons is initiated by either oxidation of an alkyl side group (26,36) or direct attack on the aromatic ring (1,9,11,12,20). Toluene has been used by a number of laboratories as a model compound to study the metabolism of aromatic hydrocarbons. When utilized as a sole carbon and energy source, toluene can be metabolized via oxidation of the methyl group to yield benzyl alcohol as the first metabolite (30,36). This type of oxidation is catalyzed by enzymes of the TOL pathway. Toluene may also be degraded by enzymes of the TOD pathway, in which initial attack is by dioxygenation of the aromatic nucleus at the 2,3 position (11,25,38). In this case, the first detectable product is (+)-cis-l(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene (cis-toluene dihydrodiol). Recently, toluene has been shown to be oxidized by whole cells of organisms which grow on toluene to o-cresol (33) and p-cresol (35a) as initial metabolites.A bacterium which grows on toluene as a sole carbon and energy source has been isolated and identified in our laboratory as Pseudomonas mendocina KR1 (31a). After growth with toluene, whole cells of this organism oxidize p-cresol at a rapid rate. We now report the separation into three protein components of the enzyme system responsible for the conversion of toluene to p-cresol. The reconstituted enzyme system catalyzed oxidation of toluene to p-cresol by an NAD(P)H-dependent monooxygenation reaction in which one atom of molecular oxygen is incorporated into the hydroxyl group of p-cresol. The reaction proceeded with a concomitant NIH shift, which suggests that toluene-3,4-oxide is the first initial oxidation product.( 6,7,8-tetrahydropterine dihydrochloride, DNase I, Bis-Tris propane, Trizma base (Tris), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonate (HEPES), morpholine propane sulfonate (MOPS), dithiothreitol (DTT), acrylamide, and N,N,N',N'-tetramethylethylenediamine were from Sigma Chemical Co., Rockford, Ill.; 2-(N-morpholine)ethanesulfate was from U.S. Biochemical Corp., Cleveland, Ohio; sodium dodecyl sulfate (SDS) was from Fisher Scientific, Fairlawn, N.J.; 2-amino-6,7 -dimethyl -...

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“…The common soil microorganism Pseudomonas putida is able to utilize ethylbenzene as a sole source of carbon and energy (Fukuda et al 1989;Gibson et al 1973). In some instances, co-oxidation or cometabolism was observed (i.e., ethylbenzene was degraded by Nocardia sp.…”

Section: Sediment and Soilmentioning

confidence: 99%

“…Biotic transformations by aerobic soil microbes involve oxidation of the ethyl side chain to form phenylacetic acid (Van der Linden and Thijsse 1965) and 1-phenylethanol (Bestetti and Galli 1984); ring hydroxylation to form 2,3-dihydroxy-1-ethylbenzene (Gibson et al 1973), 2-hydroxyphenlacetic acid, 4-hydroxyphenylacetic acid, and 2,5-and 3,4-dihydroxyphenylacetic acid (Van der Linden and Thijsse 1965); and ultimate ring cleavage to form straight chain carboxylic acids such as fumaric and acetoacetic acids (Van der Linden and Thijsse 1965). The major degradation pathways for ethylbenzene are summarized in Figure 6-3.…”

Section: Sediment and Soilmentioning

confidence: 99%

Toxicological Profile for Ethylbenzene

2002

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Initial reactions in the oxidation of ethylbenzene by Pseudomonas putida

Cited by 133 publications

References 20 publications

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

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

Toluene-4-monooxygenase, a three-component enzyme system that catalyzes the oxidation of toluene to p-cresol in Pseudomonas mendocina KR1

Toxicological Profile for Ethylbenzene

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