A novel toluene-3-monooxygenase pathway cloned from Pseudomonas pickettii PKO1

Olsen, Ronald H.; Kukor, Jerome J.; Kaphammer, Bryan

doi:10.1128/jb.176.12.3749-3756.1994

Cited by 166 publications

(133 citation statements)

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“…Five different metabolic pathways for the metabolism of toluene in aerobic microorganisms have been defined (7,16,19,24,25,27,29). When the aromatic ring is oxidized by toluene dioxygenase, cis,cis-diol is produced, which is further oxidized to 3-methylcatechol.…”

Section: Resultsmentioning

confidence: 99%

“…Different toluene monooxygenases are involved in the other four pathways. The product of toluene oxidation is benzyl alcohol, which is further oxidized to catechol (27) or o-, m-, or p-cresol according to the specificity of each enzyme (16,19,29). P. putida DOT-T1 was not able to grow on m-xylene, benzyl alcohol, m-methylbenzyl alcohol, or m-methylbenzoate, and, because the strain grown on toluene did not exhibit catechol 2,3-dioxygenase activity against catechol, this was taken as evidence that metabolism of toluene did not occur through the oxidation of the lateral chain.…”

Section: Resultsmentioning

confidence: 99%

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Isolation and expansion of the catabolic potential of a Pseudomonas putida strain able to grow in the presence of high concentrations of aromatic hydrocarbons

et al. 1995

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Pseudomonas putida DOT-T1 was isolated after enrichment on minimal medium with 1% (vol/vol) toluene as the sole C source. The strain was able to grow in the presence of 90% (vol/vol) toluene and was tolerant to organic solvents whose log P ow (octanol/water partition coefficient) was higher than 2.3. Solvent tolerance was inducible, as bacteria grown in the absence of toluene required an adaptation period before growth restarted. Mg 2؉ ions in the culture medium improved solvent tolerance. Electron micrographs showed that cells growing on high concentrations of toluene exhibited a wider periplasmic space than cells growing in the absence of toluene and preserved the outer membrane integrity. Polarographic studies and the accumulation of pathway intermediates showed that the strain used the toluene-4-monooxygenase pathway to catabolyze toluene. Although the strain also thrived in high concentrations of m-and p-xylene, these hydrocarbons could not be used as the sole C source for growth. The catabolic potential of the isolate was expanded to include m-and p-xylene and related hydrocarbons by transfer of the TOL plasmid pWW0-Km.Reports of the toxicity of aromatic solvents to microorganisms first appeared early in this century (reviewed in reference 21). Aromatic hydrocarbons become toxic when they are partitioned into lipid bilayer membranes, leading to significant changes in the structure and functioning of membrane components, e.g., disruption of the membrane potential, removal of lipids and proteins, and loss of Mg 2ϩ and Ca 2ϩ cations as well as other small molecules (21). Although a number of microbes able to grow at the expense of aromatic compounds have been isolated, their addition to the culture medium, in general, prevented growth and bacteria were able to survive only when these compounds were supplied in the vapor phase (7,27).There is considerable interest in the isolation of microbes able to thrive in high concentrations of organic solvents, because these microbes can be used as vehicles for the elimination of low-molecular-weight aromatic compounds such as toluene, styrene, benzene, and xylenes, the removal of which is of high priority (14). Furthermore, these aromatic hydrocarbons can be converted into value-added compounds such as cisdiols, epoxides, and indigo, among others (6,7,15,26). Their current synthesis by biological means requires large amounts of water, a major cost in the fermentation industry (10). Therefore, synthesis in double-phase fermentors could be more economical. Lastly, understanding of the mechanisms of solvent tolerance can be exploited in the future to generate microbes with enhanced biocatalytic potential. Inoue et al. (12,13) reported that certain Pseudomonas strains were able to thrive in the presence of more than 50% (vol/vol) toluene, although these strains were not able to grow with the aromatic hydrocarbon alone and other sources of carbon and energy were required. Cruden et al. (3) and Weber et al. (23) reported the isolation of bacterial strains able to grow at th...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Isolation and expansion of the catabolic potential of a Pseudomonas putida strain able to grow in the presence of high concentrations of aromatic hydrocarbons

et al. 1995

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“…5). These peptides are from the enzyme toluene-3-monooxygenase of the two very closely related bacteria strains Burkolderia cepacia and Ralstonia pickettii [25][26][27][28]. Both bacteria are ubiquitous in the environment but rarely cause symptomatic infection in healthy individuals [29].…”

Section: S a R W N T P T A M32253mentioning

confidence: 99%

Identification of peptides from human pathogens able to cross‐activate an HIV‐1‐gag‐specific CD4⁺ T cell clone

Venturini¹,

Allicotti²,

Zhao³

et al. 2005

Eur J Immunol

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Antigen recognition by T cells is degenerate both at the MHC and the TCR level. In this study, we analyzed the cross‐reactivity of a human HIV‐1 gag p24‐specific CD4+ T cell clone obtained from an HIV‐1‐seronegative donor using a positional scanning synthetic combinatorial peptide library (PS‐SCL)‐based biometrical analysis. A number of decapeptides able to activate the HIV‐1 gag‐specific clone were identified and shown to correspond to sequences found in other human pathogens. Two of these peptides activated the T cell clone with the same stimulatory potency as the original HIV‐1 gag p24 peptide. These findings show that an HIV‐1‐specific human T helper clone can react efficiently with peptides from other pathogens and suggest that cellular immune responses identified as being specific for one human pathogen (HIV‐1) could arise from exposure to other pathogens.

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“…Olsen and coworkers initially isolated the T3MO operon from the R. pickettii PKO1 chromosome and cloned it into plasmid pRO1966 (21). Pseudomonas aeruginosa PAO1 cells containing this plasmid were reported to form m-cresol from toluene oxidation and were reported to not convert the cresol to methylcatechol (21).…”

mentioning

confidence: 99%

“…Toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 hydroxylates the benzene ring at the ortho position to form ocresol, which is further oxidized to 3-methylcatechol (20,29). Toluene 3-monooxygenase (T3MO) of Ralstonia pickettii PKO1 was reported to hydroxylate toluene at the meta position (21), resulting in m-cresol, and toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 is specific for para hydroxylations, producing primarily p-cresol (35).…”

mentioning

confidence: 99%

Toluene 3-Monooxygenase of Ralstonia pickettii PKO1 Is a para -Hydroxylating Enzyme

2004

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Oxygenases are promising biocatalysts for performing selective hydroxylations not accessible by chemical methods. Whereas toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 hydroxylates monosubstituted benzenes at the para position and toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 hydroxylates at the ortho position, toluene 3-monooxygenase (T3MO) of Ralstonia pickettii PKO1 was reported previously to hydroxylate toluene at the meta position, producing primarily m-cresol (R. H. Olsen, J. J. Kukor, and B. Kaphammer, J. Bacteriol. 176:3749-3756, 1994). Using gas chromatography, we have discovered that T3MO hydroxylates monosubstituted benzenes predominantly at the para position. TG1/pBS(Kan)T3MO cells expressing T3MO oxidized toluene at a maximal rate of 11.5 ؎ 0.33 nmol/min/mg of protein with an apparent K m value of 250 M and produced 90% p-cresol and 10% m-cresol. This product mixture was successively transformed to 4-methylcatechol. T4MO, in comparison, produces 97% p-cresol and 3% m-cresol. Pseudomonas aeruginosa PAO1 harboring pRO1966 (the original T3MO-bearing plasmid) also exhibited the same product distribution as that of TG1/pBS(Kan)T3MO. TG1/pBS(Kan)T3MO produced 66% p-nitrophenol and 34% m-nitrophenol from nitrobenzene and 100% p-methoxyphenol from methoxybenzene, as well as 62% 1-naphthol and 38% 2-naphthol from naphthalene; similar results were found with TG1/pBS(Kan)T4MO. Sequencing of the tbu locus from pBS(Kan)T3MO and pRO1966 revealed complete identity between the two, thus eliminating any possible cloning errors. 1 H nuclear magnetic resonance analysis confirmed the structural identity of p-cresol in samples containing the product of hydroxylation of toluene by pBS(Kan)T3MO.Monooxygenases catalyze the introduction of an oxygen atom into a substrate while the second oxygen atom is reduced to water with electrons from NAD(P)H. This complex reaction often requires a metal center, and electron transfer from the reduced cofactors is mediated by additional proteins (15,17). Oxidizing enzymes are of great potential to the chemical and pharmaceutical industries due to their high regio-, chemo-, and stereoselectivity and their ability to facilitate reactions with chemically stable substrates (1, 2). Due to their complexity, biological oxidation reactions are often performed using growing or resting cells (17).One important group of bacterial oxygenases includes the toluene monooxygenases, which have additional potential applications in bioremediation (23,30,32). The aerobic biodegradation of toluene is well studied, and the pathway is available at the University of Minnesota databank (9). Xylene monooxygenase of Pseudomonas putida mt-2 hydroxylates toluene at the methyl side chain, resulting in benzyl alcohol (1). Toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 hydroxylates the benzene ring at the ortho position to form ocresol, which is further oxidized to 3-methylcatechol (20, 29). Toluene 3-monooxygenase (T3MO) of Ralstonia pickettii PKO1 was reported to hydroxylate...

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A novel toluene-3-monooxygenase pathway cloned from Pseudomonas pickettii PKO1

Cited by 166 publications

References 28 publications

Isolation and expansion of the catabolic potential of a Pseudomonas putida strain able to grow in the presence of high concentrations of aromatic hydrocarbons

Isolation and expansion of the catabolic potential of a Pseudomonas putida strain able to grow in the presence of high concentrations of aromatic hydrocarbons

Identification of peptides from human pathogens able to cross‐activate an HIV‐1‐gag‐specific CD4⁺ T cell clone

Toluene 3-Monooxygenase of Ralstonia pickettii PKO1 Is a para -Hydroxylating Enzyme

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A novel toluene-3-monooxygenase pathway cloned from Pseudomonas pickettii PKO1

Cited by 166 publications

References 28 publications

Isolation and expansion of the catabolic potential of a Pseudomonas putida strain able to grow in the presence of high concentrations of aromatic hydrocarbons

Isolation and expansion of the catabolic potential of a Pseudomonas putida strain able to grow in the presence of high concentrations of aromatic hydrocarbons

Identification of peptides from human pathogens able to cross‐activate an HIV‐1‐gag‐specific CD4+ T cell clone

Toluene 3-Monooxygenase of Ralstonia pickettii PKO1 Is a para -Hydroxylating Enzyme

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Identification of peptides from human pathogens able to cross‐activate an HIV‐1‐gag‐specific CD4⁺ T cell clone