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Journal of Bacteriology, November 1998, p. 5520-5528, Vol. 180, No. 21
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Identification of Chlorobenzene Dioxygenase Sequence Elements Involved in Dechlorination of 1,2,4,5-Tetrachlorobenzene

Stefan Beil,1 Jeremy R. Mason,2 Kenneth N. Timmis,1 and Dietmar H. Pieper1,*

Division of Microbiology, GBF-National Research Centre for Biotechnology, D-38124 Braunschweig, Germany,1 and Molecular Microbiology Group, Division of Life Sciences, King's College, University of London, London W8 7AH, United Kingdom2

Received 5 June 1998/Accepted 27 August 1998

The TecA chlorobenzene dioxygenase and the TodCBA toluene dioxygenase exhibit substantial sequence similarity yet have different substrate specificities. Escherichia coli cells producing recombinant TecA enzyme dioxygenate and simultaneously eliminate a halogen substituent from 1,2,4,5-tetrachlorobenzene but show no activity toward benzene, whereas those producing TodCBA dioxygenate benzene but not tetrachlorobenzene. A hybrid TecA dioxygenase variant containing the large alpha -subunit of the TodCBA dioxygenase exhibited a TodCBA dioxygenase specificity. Acquisition of dehalogenase activity was achieved by replacement of specific todC1 alpha -subunit subsequences by equivalent sequences of the tecA1 alpha -subunit. Substrate transformation specificities and rates by E. coli resting cells expressing hybrid systems were analyzed by high-performance liquid chromatography. This allowed the identification of both a single amino acid and potentially interacting regions required for dechlorination of tetrachlorobenzene. Hybrids with extended substrate ranges were generated that exhibited activity toward both benzene and tetrachlorobenzene. The regions determining substrate specificity in (chloro)benzene dioxygenases appear to be different from those previously identified in biphenyl dioxygenases.

* Corresponding author. Mailing address: Bereich Mikrobiologie, Gesellschaft für Biotechnologische Forschung mbH, Mascheroder Weg 1, D-38124 Braunschweig, Germany. Phone: 49-(0)531-6181-467. Fax: 49-(0)531-6181-411. E-mail: dpi{at}gbf.de.

Journal of Bacteriology, November 1998, p. 5520-5528, Vol. 180, No. 21
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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