Functional Analyses of Bph-Tod Hybrid Dioxygenase, Which Exhibits High Degradation Activity toward Trichloroethylene

Abstract

Biphenyl dioxygenase (BphDox) in Pseudomonas pseudoalcaligenes KF707 is a multicomponent enzyme consisting of an iron-sulfur protein (ISP) that is composed of α (BphA1) and β (BphA2) subunits, a ferredoxin (FD BphA3), and a ferredoxin reductase (FDRBphA4). A recombinant Escherichia coli strain expressing hybrid Dox that had replaced BphA1 with TodC1 (α 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 K m value of the hybrid Dox for TCE was 130 μM, which was lower than 250 μM for TolDox. These results suggest that the α subunit of ISP is crucial for the determination of substrate specificity and that the change in the α subunit conformation of ISP from α 2β2 to α3β3 results in the acquisition of higher affinity to TCE, which may lead to high TCE degradation activity.