Purification and properties of NADH/NADPH‐dependent p‐hydroxybenzoate hydroxylase from Corynebacterium cyclohexanicum

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Abstract
Crude soluble extracts of C. cyclohexanicum grown on cyclohexanecarboxylic acid, were found to contain 4-hydroxybenzoate-3-hydroxylase which functions with NADH and NADPH. The purified enzyme preparation was electrophoretically homogeneous and contained FAD as a prosthetic group. The relative MW of the enzyme was estimated to be .apprx. 47,000 by native and denaturated acrylamide gel electrophoresis, indicating that it is monomeric. The enzyme was stable at 60.degree. C for 10 min. The enzyme was highly specific for p-hydroxybenzoate. The activity was inhibited by several aromatic analogs of p-hydroxybenzoate such as p-aminobenzoate, p-fluorobenzoate, o-hydroxybenzoate, m-hydroxybenzoate, 2,4-dihydroxybenzoate and 2,5-dihydroxybenzoate. The Km for NADH was fairly constant, .apprx. 45 .mu.M, in the pH range 7.0-8.4; the Km for NADPH increased from 63 .mu.M to 170 .mu.M as the pH rose from 7.0 to 8.4. Velocities in the same pH range, however, were approximately constant in both cases; .apprx. 30 .mu.mol min-1 mg-1 for NADH, and 26 .mu.mol min-1 mg-1 for NADPH. Mg2+ was required for full activity of the enzyme in low concentrations of phosphate buffer. The enzyme was inhibited by Cl- which was non-competitive with respect to NADH, NADPH and p-hydroxybenzoate.