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Journal of Bacteriology, May 2000, p. 2753-2760, Vol. 182, No. 10
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

2-Hydroxycyclohexanecarboxyl Coenzyme A Dehydrogenase, an Enzyme Characteristic of the Anaerobic Benzoate Degradation Pathway Used by Rhodopseudomonas palustris

Dale A. Pelletierdagger and Caroline S. Harwood*

Department of Microbiology and Center for Biocatalysis and Bioprocessing, The University of Iowa, Iowa City, Iowa 52242

Received 18 November 1999/Accepted 29 February 2000

A gene, badH, whose predicted product is a member of the short-chain dehydrogenase/reductase family of enzymes, was recently discovered during studies of anaerobic benzoate degradation by the photoheterotrophic bacterium Rhodopseudomonas palustris. Purified histidine-tagged BadH protein catalyzed the oxidation of 2-hydroxycyclohexanecarboxyl coenzyme A (2-hydroxychc-CoA) to 2-ketocyclohexanecarboxyl-CoA. These compounds are proposed intermediates of a series of three reactions that are shared by the pathways of cyclohexanecarboxylate and benzoate degradation used by R. palustris. The 2-hydroxychc-CoA dehydrogenase activity encoded by badH was dependent on the presence of NAD+; no activity was detected with NADP+ as a cofactor. The dehydrogenase activity was not sensitive to oxygen. The enzyme has apparent Km values of 10 and 200 µM for 2-hydroxychc-CoA and NAD+, respectively. Western blot analysis with antisera raised against purified His-BadH identified a 27-kDa protein that was present in benzoate- and cyclohexanecarboxylate-grown but not in succinate-grown R. palustris cell extracts. The active form of the enzyme is a homotetramer. badH was determined to be the first gene in an operon, termed the cyclohexanecarboxylate degradation operon, containing genes required for both benzoate and cyclohexanecarboxylate degradation. A nonpolar R. palustris badH mutant was unable to grow on benzoate or cyclohexanecarboxylate but had wild-type growth rates on succinate. Cells blocked in expression of the entire cyclohexanecarboxylate degradation operon excreted cyclohex-1-ene-1-carboxylate into the growth medium when given benzoate. This confirms that cyclohex-1-ene-1-carboxyl-CoA is an intermediate of anaerobic benzoate degradation by R. palustris. This compound had previously been shown not to be formed by Thauera aromatica, a denitrifying bacterium that degrades benzoate by a pathway that is slightly different from the R. palustris pathway. 2-Hydroxychc-CoA dehydrogenase does not participate in anaerobic benzoate degradation by T. aromatica and thus may serve as a useful indicator of an R. palustris-type benzoate degradation pathway.

* Corresponding author. Mailing address: Department of Microbiology, University of Iowa, Iowa City, IA 52242. Phone: (319) 335-7783. Fax: (319) 335-7679. E-mail: caroline-harwood{at}uiowa.edu.

dagger Present address: Civil and Environmental Engineering, Stanford University, Stanford, CA 94305.

Journal of Bacteriology, May 2000, p. 2753-2760, Vol. 182, No. 10
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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