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.

and
Division of Microbiology, GBF
German
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.
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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