Cyclohexa-1,5-diene-1-carboxyl-CoA hydratase, and enzyme involved in anaerobic metabolism of benzoyl-CoA in the denitrifying bacterium Thauera aromatica

Abstract

Many aromatic compounds can be metabolized by bacteria under anoxic conditions via benzoyl-CoA as the common intermediate. The central pathway of benzoyl-CoA metabolism is initiated by-an ATP-driven reduction of the aromatic ring producing cyclohexa-1,5-diene-1-carboxyl-CoA. The 1,5-dienoyl CoA intermediate is thought to be transformed to B-hydroxycyclohex-1-ene-1-carboxyl-CoA by a specific dienoyl-CoA hydratase catalyzing the formal addition of water to one of the double bonds. This dienoyl-CoA hydratase was detected in the denitrifying bacterium Thauera aromatica after anaerobic growth with benzoate. Substrate and product were confirmed and a convenient spectrophotometric assay was developed. The equilibrium concentrations of substrate and product were almost equal. Enzyme activity was induced after anoxic growth with benzoate, in contrast to acetate. The enzyme of 28 kDa was purified from T. aromatica 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 specific activity of the enzyme in cell extract provide evidence that dienoyl-CoA hydratase is the second enzyme of the central benzoyl-CoA pathway of anaerobic aromatic metabolism in T. aromatica. Extracts of Rhodopseunomonas palustris contained high activity of cyclohex-1-ene-1-carboxyl-CoA hydratase, but no 1,5-dienoyl-CoA hydratase activity. It appears that a variant of the benzoyl-CoA pathway is operating in R. palustris in which hydration of the 1,5-dienoyl-CoA does not take place. Rather, cyclohex-1-ene-1-carboxyl-CoA is hydrated to 2-hydroxycyclohexane-1-carboxyl-CoA.