Emergence of multifunctional oxygenase activities by random priming recombination.
Biphenyl dioxygenase (Bph Dox) is responsible for the initial dioxygenation of biphenyl. The large subunit (BphA1) of Bph Dox plays a crucial role in determination of substrate specificity of biphenyl-related compounds including polychlorinated biphenyls (PCBs). Functional evolution of Bph Dox of Pseudomonas pseudoalcaligenes KF707 was accomplished by random priming recombination of the bphA1 gene, involving two rounds of in vitro recombination and mutation followed by selection for increased activity in vivo. Evolved Bph Dox acquired novel and multifunctional degradation capabilities not only for PCBs but also for dibenzofuran, dibenzo-p-dioxin, dibenzothiophene, and fluorene, the compounds scarcely attacked by the original KF707 Bph Dox. The modes of oxygenation were angular and lateral dioxygenation for dibenzofuran and dibenzo-p-dioxin, sulfoxidation for dibenzothiophene, and mono-oxygenation for fluorene. These enzymes also exhibited enhanced degradation abilities for PCB congeners, retaining 2,3-dioxygenase activity and gaining 3,4-dioxygenase activity, depending on the chlorine substitution of PCB congeners. Further mutation analysis revealed that the amino acid at position 376 in BphA1 is significantly involved in the acquisition of multifunctional oxygenase activities and mode of oxygenation.
Suenaga
H
H
Laboratory of Applied Microbiology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Hakozaki, Fukuoka 812-8581, Japan.
Goto
M
M
Furukawa
K
K
D001483:Base Sequence; D001572:Benzofurans / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers; D004147:Dioxins / Q000378:metabolism; D005449:Fluorenes / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism*; D011078:Polychlorinated Biphenyls / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology; D011995:Recombination, Genetic; D013876:Thiophenes / Q000378:metabolism
D001572:Benzofurans; D017931:DNA Primers; D004147:Dioxins; D005449:Fluorenes; D013876:Thiophenes; C041509:fluorene; C023614:dibenzofuran; D011078:Polychlorinated Biphenyls; D010105:Oxygenases; C020887:dibenzo(1,4)dioxin; C016366:dibenzothiophene