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Journal of Bacteriology, February 2001, p. 997-1011, Vol. 183, No. 3
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.3.997-1011.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Chlorocatechols Substituted at Positions 4 and 5 Are Substrates of the Broad-Spectrum Chlorocatechol 1,2-Dioxygenase of Pseudomonas chlororaphis RW71

Thomas Potrawfke, Jean Armengaud, and Rolf-Michael Wittich^*

Division of Microbiology, GBFGerman Research Centre for Biotechnology, D-38124 Braunschweig, Germany

Received 7 June 2000/Accepted 4 November 2000

The nucleotide sequence of a 10,528-bp region comprising the chlorocatechol pathway gene cluster tetRtetCDEF of the 1,2,3,4-tetrachlorobenzene via the tetrachlorocatechol-mineralizing bacterium Pseudomonas chlororaphis RW71 (T. Potrawfke, K. N. Timmis, and R.-M. Wittich, Appl. Environ. Microbiol. 64:3798-3806, 1998) was analyzed. The chlorocatechol 1,2-dioxygenase gene tetC was cloned and overexpressed in Escherichia coli. The recombinant gene product was purified, and the ,-homodimeric TetC was characterized. Electron paramagnetic resonance measurements confirmed the presence of a high-spin-state Fe(III) atom per monomer in the holoprotein. The productive transformation by purified TetC of chlorocatechols bearing chlorine atoms in positions 4 and 5 provided strong evidence for a significantly broadened substrate spectrum of this dioxygenase compared with other chlorocatechol dioxygenases. The conversion of 4,5-dichloro- or tetrachlorocatechol, in the presence of catechol, displayed strong competitive inhibition of catechol turnover. 3-Chlorocatechol, however, was simultaneously transformed, with a rate similar to that of the 4,5-halogenated catechols, indicating similar specificity constants. These novel characteristics of TetC thus differ significantly from results obtained from hitherto analyzed catechol 1,2-dioxygenases and chlorocatechol 1,2-dioxygenases.

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^* Corresponding author. Mailing address: PD TU-BS, Holbeinstrasse 24, D-38106 Braunschweig, Germany. Phone and fax: 49-(0)531-34 01 44. E-mail: rolf-michael.wittich{at}t-online.de.

Present address: BioWhittaker Europe, Parc Industriel de Petit-Rechain, B-4800 Verviers, Belgium.

Present address: Institut de Biologie Structurale, IBS-LSMP, F-38027 Grenoble Cedex 1, France.

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Journal of Bacteriology, February 2001, p. 997-1011, Vol. 183, No. 3
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.3.997-1011.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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