Crystallization and Properties of p-Hydroxybenzoate Hydroxylase from Pseudomonas putida

Abstract

Abstract 1. The inducible p-hydroxybenzoate hydroxylase of Pseudomonas putida which catalyzes the hydroxylation of p-hydroxybenzoate to protocatechuate has been obtained in crystalline form as a protein homogeneous upon ultracentrifugation and electrophoresis. The molecular weight is estimated to be 83,600. 2. The enzyme contains approximately 1 mole of flavin adenine dinucleotide per mole of protein. Reduced nicotinamide adenine dinucleotide phosphate, but not reduced nicotinamide adenine dinucleotide, serves as the hydrogen donor. 3. The enzyme is highly specific for p-hydroxybenzoate. Only four other aromatic compounds could be attacked at rates of less than 5% of the rate with p-hydroxybenzoate in all cases. 4. Under anaerobic conditions, the enzyme can catalyze the reduction of FAD by NADPH. This reaction requires the presence of the aromatic substrate to proceed at a significant rate. 5. The absorption spectrum of the enzyme in the visible region is slightly modified by the presence of the aromatic substrate.