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Toluene 3-Monooxygenase of Ralstonia pickettii PKO1 Is a para-Hydroxylating Enzyme
- Publication Year :
- 2004
- Publisher :
- American Society for Microbiology, 2004.
-
Abstract
- 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 μM 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.
- Subjects :
- Magnetic Resonance Spectroscopy
biology
Stereochemistry
Ralstonia pickettii
Nuclear magnetic resonance spectroscopy
Ralstonia
Monooxygenase
biology.organism_classification
Hydroxylation
Microbiology
Toluene
Enzymes and Proteins
chemistry.chemical_compound
Burkholderia
Biochemistry
chemistry
Oxygenases
Molecular Biology
Oxidation-Reduction
Pseudomonas mendocina
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Accession number :
- edsair.doi.dedup.....64271b0fe741e2b062cd94a198418c5c