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J Bacteriol, May 1998, p. 2330-2336, Vol. 180, No. 9
Department of Microbiology and Center for
Biocatalysis and Bioprocessing, The University of Iowa, Iowa City,
Iowa 52242
Received 7 January 1998/Accepted 6 March 1998
2-Ketocyclohexanecarboxyl coenzyme A (2-ketochc-CoA) hydrolase has
been proposed to catalyze an unusual hydrolytic ring cleavage reaction
as the last unique step in the pathway of anaerobic benzoate degradation by bacteria. This enzyme was purified from the phototrophic bacterium Rhodopseudomonas palustris by sequential
Q-Sepharose, phenyl-Sepharose, gel filtration, and hydroxyapatite
chromatography. The sequence of the 25 N-terminal amino acids of the
purified hydrolase was identical to the deduced amino acid sequence of the badI gene, which is located in a cluster of genes
involved in anaerobic degradation of aromatic acids. The deduced amino acid sequence of badI indicates that 2-ketochc-CoA
hydrolase is a member of the crotonase superfamily of proteins.
Purified BadI had a molecular mass of 35 kDa as determined by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis and a native
molecular mass of 134 kDa as determined by gel filtration. This
indicates that the native form of the enzyme is a homotetramer. The
purified enzyme was insensitive to oxygen and catalyzed the hydration
of 2-ketochc-CoA to yield pimelyl-CoA with a specific activity of 9.7 µmol min
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
2-Ketocyclohexanecarboxyl Coenzyme A Hydrolase, the
Ring Cleavage Enzyme Required for Anaerobic Benzoate Degradation by
Rhodopseudomonas palustris
1 mg of protein
1. Immunoblot
analysis using polyclonal antiserum raised against the purified
hydrolase showed that the synthesis of BadI is induced by growth on
benzoate and other proposed benzoate pathway intermediates but not by
growth on pimelate or succinate. An R. palustris mutant, carrying a chromosomal disruption of badI, did not grow
with benzoate and other proposed benzoate pathway intermediates but had
wild-type doubling times on pimelate and succinate. These data
demonstrate that BadI, the 2-ketochc-CoA hydrolase, is essential for
anaerobic benzoate metabolism by R. palustris.
*
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.
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