Monitoring trichloroethylene mineralization by Pseudomonas cepacia G4 PR1

Luu, Phillip; Yung, C. W.; Sun, Adam K.; Wood, Thomas K.

doi:10.1007/bf00164512

Cited by 35 publications

(15 citation statements)

References 13 publications

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“…TOM is a three-component, diiron enzyme encoded by the Burkholderia cepacia G4 genes tomA012345 (20), catalyzing hydroxylation of toluene to form 3-methylcatechol through the intermediate o-cresol (21). TOM also oxidizes TCE primarily to Cl Ϫ and CO 2 in vivo (22,23) and aerobically degrades various other chlorinated ethenes (20,24,25). TOM-Green originated from the first DNA shuffling of a non-heme monooxygenase and has enhanced activity for both TCE degradation and naphthalene oxidation due to a single amino acid substitution, V106A, in TomA3 (26).…”

Section: (6-fold)mentioning

confidence: 99%

Active Site Engineering of the Epoxide Hydrolase from Agrobacterium radiobacter AD1 to Enhance Aerobic Mineralization of cis-1,2-Dichloroethylene in Cells Expressing an Evolved Toluene ortho-Monooxygenase

Rui

Chen

et al. 2004

Journal of Biological Chemistry

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Chlorinated ethenes are the most prevalent groundwater pollutants, and the toxic epoxides generated during their aerobic biodegradation limit the extent of transformation. Hydrolysis of the toxic epoxide by epoxide hydrolases represents the major biological detoxification strategy; however, chlorinated epoxyethanes are not accepted by known bacterial epoxide hydrolases. Here, the epoxide hydrolase from Agrobacterium radiobacter AD1 (EchA), which enables growth on epichlorohydrin, was tuned to accept cis-1,2-dichloroepoxyethane as a substrate by accumulating beneficial mutations from three rounds of saturation mutagenesis at three selected active site residues, Phe-108, Ile-219, and Cys-248 (no beneficial mutations were found at position Ile-111). The EchA F108L/I219L/C248I variant coexpressed with a DNA-shuffled toluene ortho-monooxygenase, which initiates attack on the chlorinated ethene, enhanced the degradation of cis-dichloroethylene (cis-DCE) an infinite extent compared with wildtype EchA at low concentrations (6.8 M) and up to 10-fold at high concentrations (540 M). EchA variants with single mutations (F108L, I219F, or C248I) enhanced cis-DCE mineralization 2.5-fold (540 M), and EchA variants with double mutations, I219L/C248I and F108L/C248I, increased cis-DCE mineralization 4-and 7-fold, respectively (540 M). For complete degradation of cis-DCE to chloride ions, the apparent V max /K m for the recombinant Escherichia coli strain expressing the EchA F108L/ I219L/C248I variant was increased over 5-fold as a result of the evolution of EchA. The EchA F108L/I219L/C248I variant also had enhanced activity for 1,2-epoxyhexane (2-fold) and the natural substrate epichlorohydrin (6-fold).

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Section: (6-fold)mentioning

confidence: 99%

Active Site Engineering of the Epoxide Hydrolase from Agrobacterium radiobacter AD1 to Enhance Aerobic Mineralization of cis-1,2-Dichloroethylene in Cells Expressing an Evolved Toluene ortho-Monooxygenase

Rui

Chen

et al. 2004

Journal of Biological Chemistry

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“…Hence, the application of biocatalytic processes for 1-naphthol production may have a significant beneficial environmental impact. TOM also has the potential for bioremediation, since B. cepacia G4 expressing this enzyme was the first strain isolated that oxidizes trichloroethylene (TCE), converting it primarily to Cl Ϫ and CO 2 in vivo (17,20). TOM also aerobically degrades other chlorinated aliphatic compounds, including chlo-roform (33,35,36).…”

mentioning

confidence: 99%

Saturation Mutagenesis of Toluene ortho -Monooxygenase of Burkholderia cepacia G4 for Enhanced 1-Naphthol Synthesis and Chloroform Degradation

Rui

Kwon

Fishman

et al. 2004

Appl Environ Microbiol

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Directed evolution of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 previously created the hydroxylase ␣-subunit (TomA3) V106A variant (TOM-Green) with increased activity for both trichloroethylene degradation (twofold enhancement) and naphthalene oxidation (six-times-higher activity). In the present study, saturation mutagenesis was performed at position A106 with Escherichia coli TG1/pBS(Kan)TOMV106A to improve TOM activity for both chloroform degradation and naphthalene oxidation. Whole cells expressing the A106E variant had two times better naphthalene-to-1-naphthol activity than the wild-type cells (V max of 9.3 versus 4.5 nmol ⅐ min ؊1 ⅐ mg of protein ؊1 and unchanged K m ), and the regiospecificity of the A106E variant was unchanged, with 98% 1-naphthol formed, as was confirmed with high-pressure liquid chromatography. The A106E variant degrades its natural substrate toluene 63% faster than wild-type TOM does (2.12 ؎ 0.07 versus 1.30 ؎ 0.06 nmol ⅐ min ؊1 ⅐ mg of protein ؊1 [mean ؎ standard deviation]) at 91 M and has a substantial decrease in regiospecificity, since o-cresol (50%), m-cresol (25%), and p-cresol (25%) are formed, in contrast to the 98% o-cresol formed by wild-type TOM. The A106E variant also has an elevated expression level compared to that of wild-type TOM, as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Another variant, the A106F variant, has 2.8-times-better chloroform degradation activity based on gas chromatography (V max of 2.61 versus 0.95 nmol ⅐ min ؊1 ⅐ mg of protein ؊1 and unchanged K m ) and chloride release (0.034 ؎ 0.002 versus 0.012 ؎ 0.001 nmol ⅐ min ؊1 ⅐ mg of protein ؊1 ). The A106F variant also was expressed at levels similar to those of wild-type TOM and 62%-better toluene oxidation activity than wild-type TOM (2.11 ؎ 0.3 versus 1.30 ؎ 0.06 nmol ⅐ min ؊1 ⅐ mg of protein ؊1 ). A shift in regiospecificity of toluene hydroxylation was also observed for the A106F variant, with o-cresol (28%), m-cresol (18%), and p-cresol (54%) being formed. Statistical analysis was used to estimate that 292 colonies must be screened for a 99% probability that all 64 codons were sampled during saturation mutagenesis.Toluene ortho-monooxygenase (TOM) is encoded by the Burkholderia cepacia G4 gene cluster tomA012345 (33) and belongs to an evolutionarily related family of nonheme diiron hydroxylases found in a wide range of bacteria (16). Examples of the family include soluble methane monooxygenase (sMMO) from methanotrophic bacteria (6, 37) as well as other toluene monooxygenases, such as toluene 4-monooxygenase from Pseudomonas mendocina KR1 (40), toluene 3-monooxygenase from Ralstonia pickettii PKO1 (23), and toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1 (1). TOM is a three-component complex consisting of a 211-kDa hydroxylase (tomA1A3A4) with two binuclear iron centers in the (␣␤␥) 2 quaternary structure, a 40-kDa NADH-oxidoreductase (tomA5), and a 10.4-kDa regulatory protein (tomA2) involved in the electron transfer between the hydroxy...

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“…Fortuitously, TOM also oxidizes naphthalene, TCE, all three dichloroethylenes, and vinyl chloride (37,39). The ability of TOM to mineralize TCE primarily to CO 2 and Cl Ϫ in vivo has made this enzyme a useful biocatalyst for bioremediation (22,26). Recent studies also indicate that strains expressing TOM can degrade mixtures of chlorinated aliphatics (41).…”

mentioning

confidence: 99%

Directed Evolution of Toluene ortho -Monooxygenase for Enhanced 1-Naphthol Synthesis and Chlorinated Ethene Degradation

et al. 2002

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Trichloroethylene (TCE) is the most frequently detected groundwater contaminant, and 1-naphthol is an important chemical manufacturing intermediate. Directed evolution was used to increase the activity of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 for both chlorinated ethenes and naphthalene oxidation. When expressed in Escherichia coli, the variant TOM-Green degraded TCE (2.5 ؎ 0.3 versus 1.39 ؎ 0.05 nmol/min/mg of protein), 1,1-dichloroethylene, and trans-dichloroethylene more rapidly. Whole cells expressing TOM-Green synthesized 1-naphthol at a rate that was six times faster than that mediated by the wild-type enzyme at a concentration of 0.1 mM (0.19 ؎ 0.03 versus 0.029 ؎ 0.004 nmol/min/mg of protein), whereas at 5 mM, the mutant enzyme was active (0.07 ؎ 0.03 nmol/min/mg of protein) in contrast to the wild-type enzyme, which had no detectable activity. The regiospecificity of TOM-Green was unchanged, with greater than 97% 1-naphthol formed. The beneficial mutation of TOM-Green is the substitution of valine to alanine in position 106 of the ␣-subunit of the hydroxylase, which appears to act as a smaller "gate" to the diiron active center. This hypothesis was supported by the ability of E. coli expressing TOM-Green to oxidize the three-ring compounds, phenanthrene, fluorene, and anthracene faster than the wild-type enzyme. These results show clearly that random, in vitro protein engineering can be used to improve a large multisubunit protein for multiple functions, including environmental restoration and green chemistry.

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Monitoring trichloroethylene mineralization by Pseudomonas cepacia G4 PR1

Cited by 35 publications

References 13 publications

Active Site Engineering of the Epoxide Hydrolase from Agrobacterium radiobacter AD1 to Enhance Aerobic Mineralization of cis-1,2-Dichloroethylene in Cells Expressing an Evolved Toluene ortho-Monooxygenase

Active Site Engineering of the Epoxide Hydrolase from Agrobacterium radiobacter AD1 to Enhance Aerobic Mineralization of cis-1,2-Dichloroethylene in Cells Expressing an Evolved Toluene ortho-Monooxygenase

Saturation Mutagenesis of Toluene ortho -Monooxygenase of Burkholderia cepacia G4 for Enhanced 1-Naphthol Synthesis and Chloroform Degradation

Directed Evolution of Toluene ortho -Monooxygenase for Enhanced 1-Naphthol Synthesis and Chlorinated Ethene Degradation

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