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Originally published In Press as doi:10.1074/jbc.M102025200 on June 4, 2001

J. Biol. Chem., Vol. 276, Issue 32, 29833-29838, August 10, 2001
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Functional Analyses of Bph-Tod Hybrid Dioxygenase, Which Exhibits High Degradation Activity toward Trichloroethylene*

Tomohiro Maeda, Yukihiro Takahashi, Hikaru Suenaga, Akiko Suyama, Masatoshi Goto, and Kensuke FurukawaDagger

From the Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan

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 (FDBphA3), and a ferredoxin reductase (FDRBphA4). 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 ISPTodC1BphA2, FDBphA3, and FDRBphA4 exhibited oxygenation activities toward biphenyl, toluene, and TCE. Native polyacrylamide gel electrophoresis followed by the Ferguson plot analyses demonstrated that ISPTodC1BphA2 and ISPBphA1A2 were present as heterohexamers, whereas ISPTodC1C2 was present as a heterotetramer. The molecular activity (k0) of the hybrid Dox for TCE was 4.1 min-1, which is comparable to that of TolDox. The Km value of the hybrid Dox for TCE was 130 µM, which was lower than 250 µM 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 2beta 2 to alpha 3beta 3 results in the acquisition of higher affinity to TCE, which may lead to high TCE degradation activity.

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Laboratory of Applied Microbiology, Dept. of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan. Tel./Fax: 81-92-642-2849; E-mail address: kfurukaw@agr.kyushu-u.ac.jp.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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