Solubilization of 4-Hydroxybenzoate-polyprenyltransferase from Cell Membrane of Pseudomonas putida and Its Properties

Abstract

The enzyme of the first step in ubiquinone biosynthesis, 4-hydroxybenzoate-polyprenyltransferase was solubilized with 0.3% Triton X-100 from a membrane fraction of Pseudomonas putida IAM 1219. The enzyme had an optimum pH at 7.0-7.2 in 50 mM imidazole buffer and the apparent Km was 1.4 × 10^-4 M for nonaprenylpyrophosphate and 0.54 × 10^-5 M for 4-hydroxybenzoate. The enzyme required magnesium ion, and was completely inhibited by Cd²⁺, Cu²⁺, Ag⁺, Hg²⁺ at 0.1 mM concentation, and was also strongly inhibited by the thiol reagents, 4-chloromercuribenzoate and N-ethylmaleimide, indicating that the transferase is a thiol enzyme. The enzyme was strongly activated by adding phospholipids extracted from the bacterial cells, of which phosphatidylethanolamine was most effective.