title: Expression and purification of the recombinant subunits of toluene/o-xylene monooxygenase and reconstitution of the active complex.
abstract: This paper describes the cloning of the genes coding for each component of the complex of toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1, their expression, purification and characterization. Moreover, the reconstitution of the active complex from the recombinant subunits has been obtained, and the functional role of each component in the electron transfer from the electron donor to molecular oxygen has been determined. The coexpression of subunits B, E and A leads to the formation of a subcomplex, named H, with a quaternary structure (BEA)2, endowed with hydroxylase activity. Tomo F component is an NADH oxidoreductase. The purified enzyme contains about 1 mol of FAD, 2 mol of iron, and 2 mol of acid labile sulfide per mol of protein, as expected for the presence of one [2Fe-2S] cluster, and exhibits a typical flavodoxin absorption spectrum. Interestingly, the sequence of the protein does not correspond to that previously predicted on the basis of DNA sequence. We have shown that this depends on minor errors in the gene sequence that we have corrected. C component is a Rieske-type ferredoxin, whose iron and acid labile sulfide content is in agreement with the presence of one [2Fe-2S] cluster. The cluster is very sensitive to oxygen damage. Mixtures of the subcomplex H and of the subunits F, C and D are able to oxidize p-cresol into 4-methylcathecol, thus demonstrating the full functionality of the recombinant subunits as purified. Finally, experimental evidence is reported which strongly support a model for the electron transfer. Subunit F is the first member of an electron transport chain which transfers electrons from NADH to C, which tunnels them to H subcomplex, and eventually to molecular oxygen.
authors.affiliation: Dipartimento di Chimica Biologica and Dipartimento di Chimica Organica e Biochimica, Università di Napoli Federico II, Italy.
authors.forename: Valeria
authors.initials: V
authors.lastname: Cafaro
authors.forename: Roberta
authors.initials: R
authors.lastname: Scognamiglio
authors.forename: Ambra
authors.initials: A
authors.lastname: Viggiani
authors.forename: Viviana
authors.initials: V
authors.lastname: Izzo
authors.forename: Irene
authors.initials: I
authors.lastname: Passaro
authors.forename: Eugenio
authors.initials: E
authors.lastname: Notomista
authors.forename: Fabrizio Dal
authors.initials: FD
authors.lastname: Piaz
authors.forename: Angela
authors.initials: A
authors.lastname: Amoresano
authors.forename: Annarita
authors.initials: A
authors.lastname: Casbarra
authors.forename: Piero
authors.initials: P
authors.lastname: Pucci
authors.forename: Alberto
authors.initials: A
authors.lastname: Di Donato
mesh_terms: D001669:Biochemical Phenomena; D001671:Biochemistry; D002850:Chromatography, Gel; D004227:Dithionite / Q000737:chemistry; D004579:Electron Transport; D004583:Electrons; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D007501:Iron / Q000378:metabolism; D013058:Mass Spectrometry; D009243:NAD / Q000737:chemistry; D010105:Oxygenases / Q000737:chemistry* / Q000302:isolation & purification*; D010957:Plasmids / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013440:Sulfides / Q000737:chemistry
chemical_list: D011994:Recombinant Proteins; D013440:Sulfides; D009243:NAD; D004227:Dithionite; D007501:Iron; D010105:Oxygenases; C114360:toluene 2-xylene monooxygenase