Abstract
Epoxide carboxylase from Xanthobacter strain Py2 catalyzes the reductant- and NAD+-dependent carboxylation of aliphatic epoxides to beta-keto acids. Epoxide carboxylase from Xanthobacter strain Py2 has been resolved from cell extracts by anion-exchange chromatography into three protein components, designated I, II, and III, that are obligately required for functional reconstitution of epoxide carboxylase activity. Component II has been purified to homogeneity on the basis of its ability to complement components I and III in restoring epoxide carboxylase activity. Purified component II had a specific activity for epoxide carboxylation of 41.8 mU x min(-1) x mg(-1) when components I and III were present at saturating levels. The biochemical properties of component II reveal that it is the flavin-containing NADPH:disulfide oxidoreductase that was recently shown by other means to be associated with epoxide degradation activity in Xanthobacter strain Py2 (J. Swaving, J. A. M. de Bont, A. Westphal, and A. Dekok, J. Bacteriol. 178:6644-6646, 1996). The rate of epoxide carboxylation was dependent on the relative concentrations of the three carboxylase components. At fixed concentrations of two of the components, epoxide carboxylation rates were saturated in a hyperbolic fashion by increasing the concentration of the third variable component. Methylepoxypropane has been characterized as a time-dependent, irreversible inactivator of epoxide carboxylase activity that is proposed to be a mechanism-based inactivator of the enzyme. The addition of component I, but not that of component II or III, to methylepoxypropane-inactivated cell extracts restored epoxide carboxylase activity, suggesting that component I contains the epoxide binding and activation sites.
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