Three hydroxylations incorporating molecular oxygen in the aerobic biosynthesis of ubiquinone in Escherichia coli

Abstract

The biosynthetic origin of the O2 of ubiquinone 8 from aerobically grown E. coli was studied by 18O labeling. An apparatus was developed which allowed the growth of cells under a defined atmosphere. Mass spectral analysis of ubiquinone 8 from cells grown under highly enriched 18O2 showed that 3 O atoms of the quinone are derived from O2. It was established that O2 is incorporated into the 2 methoxyl groups (at C-5 and C-6) and 1 of the carbonyl positions of the ubiquinone molecule by demonstrating that only 1 of the incorporated O2 will exchange with water under acidic conditions that specifically catalyze the exchange of carbonyl, but not methoxyl, O2. That the C-4 carbonyl O is derived from O2 was shown by the incorporation of 3 atoms of 18O2 into ubiquinone 8 biosynthesized from added 4-hydroxybenzoic acid. Comparison of ubiquinone 8 and menaquinone 8 from E. coli grown under 18O2 confirmed that the labeled carbonyl O of the [18O2] ubiquinone 8 is incorporated biosynthetically and not by chemical exchange in the cell. The 3 hydroxylation reactions involved in the pathway for the aerobic biosynthesis of ubiquinone are apparently all catalyzed by monooxygenases. The implications of this study for the anaerobic biosynthesis of ubiquinone 8 in E. coli are discussed.