Biosynthesis of Ubiquinone Compounds with Conjugated Prenyl Side Chains

Abstract

ABSTRACT Enzymatic steps from two different biosynthetic pathways were combined in Escherichia coli , directing the synthesis of a new class of biomolecules—ubiquinones with prenyl side chains containing conjugated double bonds. This was achieved by the activity of a C 30 carotenoid desaturase, CrtN, from Staphylococcus aureus , which exhibited an inherent flexibility in substrate recognition compared to other carotenoid desaturases. By utilizing the known plasticity of E. coli 's native ubiquinone biosynthesis pathway and the unusual activity of CrtN, modified ubiquinone structures with prenyl side chains containing conjugated double bonds were generated. The side chains of the new structures were confirmed to have different degrees of desaturation by mass spectrometry and nuclear magnetic resonance analysis. In vivo 14 C labeling and in vitro activity studies showed that CrtN desaturates octaprenyl diphosphates but not the ubiquinone compounds directly. Antioxidant properties of conjugated side chain ubiquinones were analyzed in an in vitro β-carotene-linoleate model system and were found to be higher than the corresponding unmodified ubiquinones. These results demonstrate that by combining pathway steps from different branches of biosynthetic networks, classes of compounds not observed in nature can be synthesized and structural motifs that are functionally important can be combined or enhanced.