title: Oxidation of benzene to phenol, catechol, and 1,2,3-trihydroxybenzene by toluene 4-monooxygenase of Pseudomonas mendocina KR1 and toluene 3-monooxygenase of Ralstonia pickettii PKO1.
abstract: Aromatic hydroxylations are important bacterial metabolic processes but are difficult to perform using traditional chemical synthesis, so to use a biological catalyst to convert the priority pollutant benzene into industrially relevant intermediates, benzene oxidation was investigated. It was discovered that toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1, toluene 3-monooxygenase (T3MO) of Ralstonia pickettii PKO1, and toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 convert benzene to phenol, catechol, and 1,2,3-trihydroxybenzene by successive hydroxylations. At a concentration of 165 microM and under the control of a constitutive lac promoter, Escherichia coli TG1/pBS(Kan)T4MO expressing T4MO formed phenol from benzene at 19 +/- 1.6 nmol/min/mg of protein, catechol from phenol at 13.6 +/- 0.3 nmol/min/mg of protein, and 1,2,3-trihydroxybenzene from catechol at 2.5 +/- 0.5nmol/min/mg of protein. The catechol and 1,2,3-trihydroxybenzene products were identified by both high-pressure liquid chromatography and mass spectrometry. When analogous plasmid constructs were used, E. coli TG1/pBS(Kan)T3MO expressing T3MO formed phenol, catechol, and 1,2,3-trihydroxybenzene at rates of 3 +/- 1, 3.1 +/- 0.3, and 0.26 +/- 0.09 nmol/min/mg of protein, respectively, and E. coli TG1/pBS(Kan)TOM expressing TOM formed 1,2,3-trihydroxybenzene at a rate of 1.7 +/- 0.3 nmol/min/mg of protein (phenol and catechol formation rates were 0.89 +/- 0.07 and 1.5 +/- 0.3 nmol/min/mg of protein, respectively). Hence, the rates of synthesis of catechol by both T3MO and T4MO and the 1,2,3-trihydroxybenzene formation rate by TOM were found to be comparable to the rates of oxidation of the natural substrate toluene for these enzymes (10.0 +/- 0.8, 4.0 +/- 0.6, and 2.4 +/- 0.3 nmol/min/mg of protein for T4MO, T3MO, and TOM, respectively, at a toluene concentration of 165 microM).
authors.affiliation: Department of Chemical Engineering, University of Connecticut, Storrs, CT 06269-3222, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Tao
authors.forename: Ayelet
authors.initials: A
authors.lastname: Fishman
authors.forename: William E
authors.initials: WE
authors.lastname: Bentley
authors.forename: Thomas K
authors.initials: TK
authors.lastname: Wood
mesh_terms: D001483:Base Sequence; D001554:Benzene / Q000378:metabolism*; D002396:Catechols / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000235:genetics / Q000502:physiology*; D019800:Phenol / Q000378:metabolism*; D044203:Pseudomonas mendocina / Q000201:enzymology*; D011748:Pyrogallol / Q000378:metabolism*; D043367:Ralstonia / Q000201:enzymology*
chemical_list: D002396:Catechols; D004269:DNA, Bacterial; D011748:Pyrogallol; D019800:Phenol; D010105:Oxygenases; C087733:toluene-3-monooxygenase; C068610:toluene-4-monooxygenase; D001554:Benzene; C034221:catechol