Vitamin K (menaquinone) biosynthesis in bacteria: high-performance liquid chromatographic assay of the overall synthesis of o-succinylbenzoic acid and of 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid synthase

An early enzyme in menaquinone (vitamin K2) biosynthesis is the synthase forming 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid (SHCHC) from isochorismic acid. In turn, SHCHC is aromatized to o-succinylbenzoic acid (OSB) by OSB synthase. An assay for the combined activity of these two enzymes ("overall OSB synthesis") has been developed using a high-performance liquid chromatographic method for the quantitation of OSB. The assay, which measures as little as 0.1 nmol of OSB, is vastly superior to the radiogas chromatographic method previously used to estimate overall OSB synthesis. To measure SHCHC synthase activity separately, the enzymatically formed SHCHC is converted nonenzymatically to OSB (heating to 80 degrees C, pH 10, 10 min), which is then quantitated by the HPLC assay. The preparation of the substrate, isochorismic acid, and its purification by preparative HPLC are also described.