CYCLOHEXA-1,5-DIENE-1-CARBOXYL-COA HYDRATASE, AN ENZYME INVOLVED IN ANAEROBIC METABOLISM OF BENZOYL-COA IN THE DENITRIFYING BACTERIUM THAUERA-AROMATICA

Many aromatic compounds can be metabolized by bacteria under anoxic co nditions via benzoyl-CoA as the common intermediate. The central pathw ay of benzoyl-CoA metabolism is initiated by an ATP-driven reduction o f the aromatic ring producing cyclohexa-1,5-diene-1-carboxyl-CoA. The 1,5-dienoyl-CoA intermediate is thought to be transformed to 6-hydroxy cyclohex-1-ene-1-carboxyl-CoA by a specific dienoyl-CoA hydratase cata lyzing the formal addition of water to one of the double bonds. This d ienoyl-CoA hydratase was detected in the denitrifying bacterium Thauer a aromatica after anaerobic growth with benzoate. Substrate and produc t were confirmed and a convenient spectrophotometric assay was develop ed. The equilibrium concentrations of substrate and product were almos t equal. Enzyme activity was induced after anoxic growth with benzoate , in contrast to acetate. The enzyme of 28 kDa was purified from T. ar omatica and was found to be highly specific for the cyclic 1,5-dienoyl -CoA. A second 29-kDa enoyl-CoA hydratase acted on crotonyl-Coa; this highly active enoyl-CoA hydratase also acted slowly on cyclohex-1-ene- 1-carboxyl-CoA. The regulation of expression of dienoyl-CoA hydratase activity, the kinetic constants, the substrate specificity, and the sp ecific activity of the enzyme in cell extract provide evidence that di enoyl-CoA hydratase is the second enzyme of the central benzoyl-CoA pa thway of anaerobic aromatic metabolism in T. aromatica. Extracts of Rh odopseudomonas palustris contained high activity of cyclohex-1-ene-1-c arboxyl-CoA hydratase, but no 1,5-dienoyl-CoA hydratase activity. It a ppears that a variant of the benzoyl-CoA pathway is operating in R. pa lustris in which hydration of the 1,5-dienoyl-CoA does not take place. Rather, cyclohex-1-ene-1-carboxyl-CoA is hydrated to 2-hydroxycyclohe xane-1-carboxyl-CoA.