2-KETOCYCLOHEXANECARBOXYL COENZYME-A HYDROLASE, THE RING-CLEAVAGE ENZYME REQUIRED FOR ANAEROBIC BENZOATE DEGRADATION BY RHODOPSEUDOMONAS-PALUSTRIS

2-Ketocyclohexanecarboxyl coenzyme A (3-ketochc-CoA) hydrolase has bee n 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-Sepharos e, 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 ac ids. The deduced amino acid sequence of badI indicates that 2-ketoche- CoA hydrolase is a member of the crotonase superfamily of proteins. Pu rified BadI had a molecular mass of 35 kDa as determined by sodium dod ecyl sulfate-polyacrylamide gel electrophoresis and a native molecular mass of 35 kDa as determined by gel filtration. This indicates that t he native form of the enzyme is a homotetramer. The purified enzyme wa s insensitive to oxygen and catalyzed the hydration of 2-ketoche-CoA t o yield pimelyl-CoA with a specific activity of 9.7 mu mol min-L mg of protein(-1). Immunoblot analysis using polyclonal antiserum raised ag ainst the purified hydrolase showed that the synthesis of BadI is indu ced by growth on benzoate and other proposed benzoate pathway intermed iates but not by growth on pimelate or succinate. An R. palustris muta nt, carrying a chromosomal disruption of badI, did not grow with benzo ate and other proposed benzoate pathway intermediates but had wild-typ e doubling times on pimelate and succinate, These data demonstrate tha t BadI, the 2-ketoche-CoA hydrolase, is essential for anaerobic benzoa te metabolism by R. palustris.