Protocatechuate 3,4-dioxygenase from Acinetobacter calcoaceticus

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Abstract
Protocatechuate 3,4-dioxygenase (PCD) from p-hydroxybenzoate-induced cells of A. calcoaceticus was purified by heat and protamine sulfate treatment, ammonium sulfate fractionation, DEAE-cellulose, and Sephadex G-200 column chromatography. The enzyme appears to be homogeneous by ultracentrifugation and acrylamide gel electrophoresis. This is the 1st report of PCD purified from Acinetobacter. For comparison, crystalline Pseudomonas PCD was also obtained. The enzymes from Acinetobacter and Pseudomonas are quite similar in their molecular weight, molecular size and Fe content. The specific enzyme activity of PCD from Acinetobacter is about 1/3 of that from Pseudomonas, despite their similar Fe content. Visible and circular dichroism spectra indicate some conformational differences between these 2 enzymes. Protocatechualdehyde, a competitive deadend inhibitor, binds Pseudomonas PCD more effectively than Acinetobacter PCD. p-Hydroxymercuribenzoate, specific for free -SH groups, inhibits only Acinetobacter PCD and shows no effect on Pseudomonas PCD. Amino acid analyses reveal very low proline and methionine content with higher lysine, glutamic acid and isoleucine compositions for Acinetobacter PCD. Other properties, including active center conformation, were studied and discussed.