Purification and properties of L-4-hydroxymandelate oxidase from Pseudomonas convexa.

An inducible membrane-bound L-4-hydroxymandelate oxidase (decarboxylating) from Pseudomonas convexa has been solubilized and partially purified. It catalyzes the conversion of L-4-hydroxymandelic acid to 4-hydroxybenzaldehyde in a single step with the stoichiometric consumption of O2 and liberation of CO2. The enzyme is optimally active at pH 6.6 and at 55 degrees C. It requires FAD and Mn2+ for its activity. The membrane-bound enzyme is more stable than the solubilized and purified enzyme. After solubilization it gradually loses its activity when kept at 5 degrees C which can be fully reactivated by freezing and thawing. The Km values for DL-4-hydroxymandelate and FAD are 0.44 mM and 0.038 mM respectively. The enzyme is highly specific for DL-4-hydroxymandelic acid. DL-3,4-Dihydroxymandelic acid competitively inhibited the enzyme reaction. From the Dixon plot the Ki for DL-3,4-dihydroxymandelic acid was calculated to be 1.8 X 10(-4) M. The enzyme is completely inactivated by thiol compounds and not affected by thiol inhibitors. The enzyme is also inhibited by denaturing agents, heavy metal ions and by chelating agents.