and Characterization of a New Isochorismate Synthase from Escherichia coli

The first committed step in the biosynthesis of menaquinone (vitamin K2) is the conversion of chorismate to isochorismate, which is mediated by an isochorismate synthase encoded by the menF gene. This isochoris-mate synthase (MenF) is distinct from the entC-encoded isochorismate synthase (EntC) involved in entero-bactin biosynthesis. MenF has been overexpressed under the influence of the T7 promoter and purified to homogeneity. The purified protein was found to have a molecular mass of 98 kDa as determined by gel filtration column chromatography on Sephacryl S-200. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis re-vealed a molecular mass of 48 kDa. Thus, the enzyme is a homodimer. The purified enzyme showed a pH optimum of 7.5 to 8.0 and a temperature optimum of 37°C. The enzyme carries out the irreversible conversion of chorismate to isochorismate in the presence of Mg21. The enzyme was found to have a Km of 195 6 23 mM and a kcat of 80 min 21. In the presence of 30 mM b-mercaptoethanol (BME), the kcat increased to 176 min 21. The reducing agents BME and dithiothreitol stimulated the enzymatic activity more than twofold. Treatment of the enzyme with the cysteine-specific modifying reagent N-ethylmaleimide (NEM) resulted in the complete loss of activity. Preincubation of the enzyme with the substrate, chorismate, before NEM treatment resulted i