J. Bacteriol., 05 1996, 2656-2661, Vol 178, No. 9
Copyright © 1996, American Society for Microbiology

NADPH-dependent reductive ortho dehalogenation of 2,4-dichlorobenzoic acid in Corynebacterium sepedonicum KZ-4 and Coryneform bacterium strainNTB-1 via 2,4-dichlorobenzoyl coenzyme A

V Romanov and RP Hausinger
Center for Microbial Ecology, Michigan State University, East Lansing 48824, USA.

Corynebacterium sepedonicum KZ-4, described earlier as a strain capable of growth on 2,4-dichlorobenzoate (G.M. Zaitsev and Y.N. Karasevich, Mikrobiologiya 54:356-369, 1985), is known to metabolize this substrate via 4-hydroxybenzoate and protocatechuate, and evidence consistent with an initial reductive dechlorination step to form 4-chlorobenzoate was found in another coryneform bacterium, strain NTB-1 (W.J.J. van den Tweel, J.B. Kok, and J.A.M. de Bont, Appl. Environ. Microbiol. 53:810- 815, 1987). 2-Chloro-4-fluorobenzoate was found to be converted stoichiometrically to 4-fluorobenzoate by resting cells of strain KZ-4, compatible with a reductive process. Experiments with cell extracts demonstrated that Mg - ATP and coenzyme A (CoA) were required to stimulate reductive dehalogenation, consistent with the intermediacy of 2-chloro-4-fluoro-benzoyl-CoA and 2,4-dichlorobenzoyl-CoA thioesters. 2,4-Dichlorobenzoyl-CoA was shown to be converted to 4-chlorobenzoyl- CoA in a novel NADPH-dependent reaction in extracts of both KZ-4 and NTB-1. In addition to the ligase and reductive dehalogenase activities, hydrolytic 4-chlorobenzoyl-CoA dehalogenase and thioesterase activities, 4-hydroxybenzoate 3-monooxygenase, and protocatechuate 3,4- dioxygenase activities were demonstrated to be present in the soluble fraction of KZ-4 extracts following ultracentrifugation. We propose that the pathway for 2,4-dichlorobenzoate catabolism in strains KZ-4 and NTB-1 involves formation of 2,4-dichlorobenzoyl-CoA, NADPH- dependent ortho dehalogenation yielding 4-chlorobenzoyl-CoA, hydrolytic removal of chlorine from the para position to generate 4-hydroxybenzoyl- CoA, hydrolysis to form 4-hydroxybenzoate, oxidation to yield protocatechuate, and oxidative ring cleavage.

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