Purification and characterization of toluene 2-monooxygenase from Burkholderia cepacia G4

Abstract

Recent in vivo studies indicate that ring monooxygenation is a widespread mechanism by which bacteria metabolize aromatic hydrocarbons and obtain carbon and energy. In this study, toluene 2-monooxygenase from Burkholderia (formerly Pseudomonas) cepacia G4 was purified to homogeneity and found to be a three-component enzyme system. The reconstituted enzyme system oxidized toluene to o-cresol and o-cresol to 3-methylcatechol, an important intermediate for growth of the bacterium on toluene. Steady-state kinetic parameters measured for the water-soluble substrate o-cresol were a Km of 0.8 microM and a Vmax of 131 nmol min-1 (mg of hydroxylase protein)-1. The three protein components were (1) a 40 kDa polypeptide containing one FAD and a [2Fe2S] cluster, (2) a 10.4 kDa polypeptide that contained no identifiable metals or organic cofactors, and (3) a 211 kDa alpha 2 beta 2 gamma 2 component containing five to six iron atoms. The 40 kDa flavo-iron-sulfur protein oxidized NADH and transferred electrons to cytochrome c, dyes, and the alpha 2 beta 2 gamma 2 component. It is analogous to other NADH oxidoreductase components found in a wide range of bacterial mono- and dioxygenases. The 10.4 kDa component, added to the other two components and NADH, increased toluene oxidation rates 10-fold. The alpha 2 beta 2 gamma 2 component was indicated to contain the site for toluene binding and hydroxylation by the following observations: (1) tight binding to a toluene affinity column; (2) oxidation of toluene after reduction of the protein with dithionite and adding O2; (3) H2O2-dependent toluene oxidation and catalase activity; and (4) spectroscopic studies of the iron atoms in the component. The alpha 2 beta 2 gamma 2 component had no significant absorbance in the visible region. EPR spectroscopy yielded a signal at g = 16 upon addition of > 2 equiv of electrons per 2 Fe atoms. Taken with the quantitation of five to six iron atoms, the data suggest that the alpha 2 beta 2 gamma 2 component contains two binuclear iron centers. In total, the structural, spectroscopic, and catalytic features of toluene 2-monooxygenase are reminiscent of soluble methane monooxygenase obtained from methanotrophic bacteria. The two enzyme systems also differ in many subtle ways; for example, they oxidize toluene with completely different regiospecificity.

tRNA-modifying MiaE protein fromSalmonella typhimuriumis a nonheme diiron monooxygenase

“…In addition to the band at 280 nm ( 280 Ϸ 60,000 M Ϫ1 cm Ϫ1 ) corresponding to protein absorption, the spectrum displays two additional bands at 320 and 370 nm associated with the presence of iron in the protein. The optical properties described here are comparable with those of the active site of the R2 subunit of aerobic ribonucleotide reductase from E. coli, phenol hydroxylase from Pseudomonas sp strain CF 600 and toluene-2-monooxygenase from Burkholderia cepacia G4 proteins known to contain -oxo-bridged diiron clusters with primary ligation sphere consisting of oxygen and nitrogen ligands (16)(17)(18). Similarity to these enzymes is further supported by EPR spectroscopy.…”

A Monooxygenase Catalyzes Sequential Dechlorinations of 2,4,6-Trichlorophenol by Oxidative and Hydrolytic Reactions

“…Toluene 2-monooxygenase of B. cepacia G4 oxidizes toluene to ocresol and then to 3-methylcatechol (25). Human cytochrome P450 2B6 monooxygenase oxidizes efavirenz, an HIV-1 reverse transcriptase inhibitor, to 8-hydroxyefavirenz and then to 8,14-dihydroxyefavirenz with similar kinetic parameters (26).…”

Molecular analysis of the soluble butane monooxygenase from ‘Pseudomonas butanovora’ a aThe GenBank accession number for the bmoXYBZDC sequence is AY093933.

“…This component was heat labile, but it was not noticeably enriched in any particular peptides. Further purification and characterization is required to confirm our supposition that this component contains the small-molecular-mass ' regulatory protein ' found in sMMOs (Fox et al, 1989 ;Green & Dalton, 1985), alkene monooxygenases (Miura & Dalton, 1995 ;Small & Ensign, 1997), and several aromatic monooxygenases (Newman & Wackett, 1995 ;.…”

“…The substrate range of sMMO includes C " -C ( alkanes, alkenes and some aromatic compounds (Green & Dalton, 1989). Soluble toluene monooxygenases have also been described, including four-component diiron monooxygenases (Pikus et al, 1996), three-component diiron monooxygenases (Newman & Wackett, 1995) and three-component mononuclear iron dioxygenases (Subramanian et al, 1979(Subramanian et al, , 1981(Subramanian et al, , 1985.…”