title: Regiospecific oxidation of naphthalene and fluorene by toluene monooxygenases and engineered toluene 4-monooxygenases of Pseudomonas mendocina KR1.
abstract: The regiospecific oxidation of the polycyclic aromatic hydrocarbons naphthalene and fluorene was examined with Escherichia coli strains expressing wildtype toluene 4-monooxygenase (T4MO) from Pseudomonas mendocina KR1, toluene para-monooxygenase (TpMO) from Ralstonia pickettii PKO1, toluene ortho-monooxygenase (TOM) from Burkholderia cepacia G4, and toluene/ortho-xylene monooxygenase (ToMO) from P. stutzeri OX1. T4MO oxidized toluene (12.1+/-0.8 nmol/min/mg protein at 109 microM), naphthalene (7.7+/-1.5 nmol/min/mg protein at 5 mM), and fluorene (0.68+/-0.04 nmol/min/mg protein at 0.2 mM) faster than the other wildtype enzymes (2-22-fold) and produced a mixture of 1-naphthol (52%) and 2-naphthol (48%) from naphthalene, which was successively transformed to a mixture of 2,3-, 2,7-, 1,7-, and 2,6-dihydroxynaphthalenes (7%, 10%, 20%, and 63%, respectively). TOM and ToMO made 1,7-dihydroxynaphthalene from 1-naphthol, and ToMO made a mixture of 2,3-, 2,6-, 2,7-, and 1,7-dihydroxynaphthalene (26%, 22%, 1%, and 44%, respectively) from 2-naphthol. TOM had no activity on 2-naphthol, and T4MO had no activity on 1-naphthol. To take advantage of the high activity of wildtype T4MO but to increase its regiospecificity on naphthalene, seven engineered enzymes containing mutations in T4MO alpha hydroxylase TmoA were examined; the selectivity for 2-naphthol by T4MO I100A, I100S, and I100G was enhanced to 88-95%, and the selectivity for 1-naphthol was enhanced to 87% and 99% by T4MO I100L and G103S/A107G, respectively, while high oxidation rates were maintained except for G103S/A107G. Therefore, the regiospecificity for naphthalene oxidation was altered to practically pure 1-naphthol or 2-naphthol. All four wildtype monooxygenases were able to oxidize fluorene to different monohydroxylated products; T4MO oxidized fluorene successively to 3-hydroxyfluorene and 3,6-dihydroxyfluorene, which was confirmed by gas chromatography-mass spectrometry and 1H nuclear magnetic resonance analysis. TOM and its variant TomA3 V106A oxidize fluorene to a mixture of 1-, 2-, 3-, and 4-hydroxyfluorene. This is the first report of using enzymes to synthesize 1-, 3-, and 4-hydroxyfluorene, and 3,6-dihydroxyfluorene from fluorene as well as 2-naphthol and 2,6-dihydroxynaphthalene from naphthalene.
authors.affiliation: Department of Chemical Engineering, University of Connecticut, Storrs, Connecticut 06269-3222, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Tao
authors.forename: William E
authors.initials: WE
authors.lastname: Bentley
authors.forename: Thomas K
authors.initials: TK
authors.lastname: Wood
mesh_terms: D001673:Biodegradation, Environmental; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005449:Fluorenes / Q000737:chemistry / Q000493:pharmacokinetics*; D009281:Naphthalenes / Q000737:chemistry / Q000493:pharmacokinetics*; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D044203:Pseudomonas mendocina / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity; D014050:Toluene / Q000737:chemistry / Q000378:metabolism
chemical_list: D005449:Fluorenes; D009281:Naphthalenes; C031721:naphthalene; D014050:Toluene; C041509:fluorene; D010105:Oxygenases; C068610:toluene-4-monooxygenase