Functional analyses of Bph-Tod hybrid dioxygenase, which exhibits high degradation activity toward trichloroethylene.
Biphenyl dioxygenase (BphDox) in Pseudomonas pseudoalcaligenes KF707 is a multicomponent enzyme consisting of an iron-sulfur protein (ISP) that is composed of alpha (BphA1) and beta (BphA2) subunits, a ferredoxin (FD(BphA3)), and a ferredoxin reductase (FDR(BphA4)). A recombinant Escherichia coli strain expressing hybrid Dox that had replaced BphA1 with TodC1 (alpha subunit of toluene dioxygenase (TolDox) of Pseudomonas putida) exhibited high activity toward trichloroethylene (TCE) (Furukawa, K., Hirose, J., Hayashida, S., and Nakamura, K. (1994) J. Bacteriol. 176, 2121-2123). In this study, ISP, FD, and FDR were purified and characterized. Reconstitution of the dioxygenase components consisting of purified ISP(TodC1BphA2), FD(BphA3), and FDR(BphA4) exhibited oxygenation activities toward biphenyl, toluene, and TCE. Native polyacrylamide gel electrophoresis followed by the Ferguson plot analyses demonstrated that ISP(TodC1BphA2) and ISP(BphA1A2) were present as heterohexamers, whereas ISP(TodC1C2) was present as a heterotetramer. The molecular activity (k(0)) of the hybrid Dox for TCE was 4.1 min(-1), which is comparable to that of TolDox. The K(m) value of the hybrid Dox for TCE was 130 microm, which was lower than 250 microm for TolDox. These results suggest that the alpha subunit of ISP is crucial for the determination of substrate specificity and that the change in the alpha subunit conformation of ISP from alpha(2)beta(2) to alpha(3)beta(3) results in the acquisition of higher affinity to TCE, which may lead to high TCE degradation activity.
Maeda
T
T
Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan.
Takahashi
Y
Y
Suenaga
H
H
Suyama
A
A
Goto
M
M
Furukawa
K
K
D004591:Electrophoresis, Polyacrylamide Gel; D005287:Ferredoxin-NADP Reductase / Q000302:isolation & purification; D005288:Ferredoxins / Q000302:isolation & purification; D007506:Iron-Sulfur Proteins / Q000302:isolation & purification; D007700:Kinetics; D008956:Models, Chemical; D010105:Oxygenases / Q000737:chemistry* / Q000502:physiology*; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011549:Pseudomonas / Q000201:enzymology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D014241:Trichloroethylene / Q000378:metabolism*; D014752:Vinyl Chloride / Q000737:chemistry
D005288:Ferredoxins; D007506:Iron-Sulfur Proteins; D011993:Recombinant Fusion Proteins; D014241:Trichloroethylene; D010105:Oxygenases; C023172:toluene dioxygenase; C074161:biphenyl-2,3-dioxygenase; D005287:Ferredoxin-NADP Reductase; D014752:Vinyl Chloride