CYCLOHEXA-1,5-DIENE-1-CARBOXYL-COA HYDRATASE, AN ENZYME INVOLVED IN ANAEROBIC METABOLISM OF BENZOYL-COA IN THE DENITRIFYING BACTERIUM THAUERA-AROMATICA
D. Laempe et al., CYCLOHEXA-1,5-DIENE-1-CARBOXYL-COA HYDRATASE, AN ENZYME INVOLVED IN ANAEROBIC METABOLISM OF BENZOYL-COA IN THE DENITRIFYING BACTERIUM THAUERA-AROMATICA, European journal of biochemistry, 255(3), 1998, pp. 618-627
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.