In Vitro Assay for the Characterization of RquA

Abstract

To survive in low‐oxygenic environments, microbial organisms employ anaerobic respiration. In certain organisms, the terpenoid quinone known as rhodoquinone (RQ) plays a key role in this process, though few details are known about how RQ itself is produced or what impact it may have on the cellular respiration process from a biomedical standpoint. Investigations into the rquA gene product (RquA), an enzyme that is required for the production of RQ in Rhodospirillum rubrum and other microbial species, and its role in anaerobic energy metabolism may provide a new target for antibacterial drug treatments through inhibition. Previous findings have shown that the in vivo expression of RquA results in the production of RQ in two species that do not naturally produce it, E. coli and yeast. RquA is responsible for the conversion of ubiquinone (Q) to RQ but it is not clear what additional cofactors are required for the conversion. Using in vitro assays with synthetic Q, purified RquA or whole cell lysate, potential amino donors, and a variety of co‐factors such as S‐adenosyl methionine, we investigated the essential chemical components to perform this conversion. With these results, potential inhibition pathways may be identified and further investigated through assays and visualization methods such as X‐ray crystallography.Support or Funding InformationNew Frontiers in Research Fund, Gonzaga Science Research Program, CURCI