Characterisation of a chromosomally encoded catechol 1,2-dioxygenase (E.C. 1.13.11.1) from Alcaligenes eutrophus CH34
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© Springer-Verlag 1996
O R I G I N A L PA P E R
Ginette Sauret-Ignazi · Jean Gagnon · Claude Béguin · Michel Barrelle · Yves Markowicz · Jean Pelmont · Ariane Toussaint
Characterisation of a chromosomally encoded catechol 1,2-dioxygenase (E.C. 1.13.11.1) from Alcaligenes eutrophus CH34
Received: 20 November 1996 / Accepted: 11 April 1996
Abstract Alcaligenes eutrophus CH34 used benzoate as a sole source of carbon and energy, degrading it through the 3-oxoadipate pathway. All the enzymes required for this degradation were shown to be encoded by chromosomal genes. Catechol 1,2-dioxygenase activity was induced by benzoate, catechol, 4-chlorocatechol, and muconate. The enzyme is most likely a homodimer, with an apparent molecular weight of 76,000 ± 500. According to several criteria, its properties are intermediate between those of catechol 1,2-dioxygenases (CatA) and chlorocatechol 1,2-dioxygenases (ClcA). The determined Km for catechol is the lowest among known catechol and chlorocatechol dioxygenases. Similar Km values were found for para-substituted catechols, although the catalytic constants were much lower. The catechol 1,2-dioxygenase from strain CH34 is unique in its property to transform tetrachlorocatechol; however, excess substrate led to a marked reversible inhibition. Some meta- and multi-substituted catechols behaved similarly. The determined Km (or Ki) values for para- or meta-substituted catechols suggest that the presence of an electron-withdrawing substituent at one of these positions results in a higher affinity of the enzyme for the ligand. Results of studies of recognition by the enzyme of various nonmetabolised aromatic compounds are also discussed.
G. Sauret-Ignazi (Y) · Y. Markowicz · J. Pelmont · A. Toussaint Laboratoire de Génétique Microbienne (DRED JE 117), Université Joseph Fourier, BP 53, F-38041 Grenoble cédex 9, France Tel. +33-7651-4346; Fax +33-7651-4336 e-mail: [email protected] J. Gagnon Laboratoire d’Enzymologie Moléculaire, Institut de Biologie Structurale (CEA-CNRS UPR 9015), 41 avenue des Martyrs, F-38021 Grenoble cédex 1, France C. Béguin · M. Barrelle Laboratoire d’Etudes Dynamiques et Structurales de la Sélectivité 2 (CNRS URA 332), Université Joseph Fourier, BP 53, F-38041 Grenoble cédex 9, France
Key words Catechol 1,2-dioxygenase · Alcaligenes eutrophus CH34 · Substituted catechol degradation · Excess substrate inhibition · Phenol interactions
Introduction Chlorinated catechols have been reported to be key intermediates in the biodegradation by aerobic organisms of various chloroaromatics such as chlorobenzoates and chlorophenoxyacetates (Reineke and Knackmuss 1988; Häggblom 1992). Cleavage of the aromatic ring in these catechols occurs by dioxygenation prior to dechlorination, and hence the first attack on these compounds produces various nonaromatic intermediates, which are dehalogenated at a later step (Reineke and Knackmuss 1988), with the removal of chlorine atoms as chloride. The complete degradation of chlorinated catechol is commonly dependent on plasmid-
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