title: Steady-state kinetic characterization of evolved biphenyl dioxygenase, which acquired novel degradation ability for benzene and toluene.
abstract: Biphenyl dioxygenase (Bph Dox) catalyzes initial oxygenation in the bacterial biphenyl degradation pathway. Bph Dox in Pseudomonas pseudoalcaligenes KF707 is a Rieske type three-component enzyme in which a large subunit (encoded by the bphA1 gene) plays an important role in the substrate specificity of Bph Dox. Steady-state kinetic assays using purified enzyme components demonstrated that KF707 Bph Dox had a kcat/Km of 33.1 x 10(3) (M(-1) s(-1)) for biphenyl. Evolved 1072 Bph Dox generated by the process of DNA shuffling (Suenaga, H. et al., J. Bacteriol., 184, 3682-3688 (2002)) exhibited enhanced degradation activity not only for biphenyl (kcat/Km of 62.2 x 10(3) [M(-1) s(-1)]) but also for benzene and toluene, compounds that are rarely attacked by KF707 Bph Dox. These results suggest that evolved 1072 Bph Dox acquires higher affinities and catalytic efficiencies for various substrates than the original KF707 enzyme.
authors.affiliation: Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan. sunega-hikaru@aist.go.jp
authors.forename: Hikaru
authors.initials: H
authors.lastname: Suenaga
authors.forename: Mika
authors.initials: M
authors.lastname: Sato
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Goto
authors.forename: Mariko
authors.initials: M
authors.lastname: Takeshita
authors.forename: Kensuke
authors.initials: K
authors.lastname: Furukawa
mesh_terms: D001554:Benzene / Q000378:metabolism*; D001665:Binding Sites; D007506:Iron-Sulfur Proteins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D044205:Pseudomonas pseudoalcaligenes / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity; D014050:Toluene / Q000378:metabolism*
chemical_list: D007506:Iron-Sulfur Proteins; D014050:Toluene; D010105:Oxygenases; D001554:Benzene