Optimizing the Purification and Investigating the Function of Rhodoquinone Biosynthesis Enzyme A

Abstract

Organisms require a constant supply of energy to survive, and they store this energy in the form of adenosine triphosphate (ATP). Cellular respiration is the major source of energy; however, in many organisms it requires oxygen to continue. Organisms that reside in oxygen deficient environments have adapted to continue ATP production by using the electron carrier rhodoquinone instead of ubiquinone. Rhodoquinone has a much lower reduction potential than ubiquinone allowing for fumarate to be reduced and the production of ATP to continue in the absence of oxygen. Rhodoquinone biosynthesis enzyme A (RquA) was the first gene discovered that was required for rhodoquinone biosynthesis. RquA requires S-adenosylmethionine and Mn2+ to catalyze the conversion of ubiquinone to rhodoquinone. In this thesis an optimal method was created to purify RquA from Euglena gracilis and isolated RquA was used for functional assays. High performance liquid chromatography was used to perform functional based assays to determine the production of rhodoquinone. The optimal condition required for the activity of RquA was determined and S-adenosyl-l-homocysteine and sinefungin were identified as weak inhibitors of RquA. The solubility of RquA was increased by creating two mutants that either deleted a predicted key α-helix or the amino acid sequence was mutated to make it more hydrophilic. This research is the first characterization of RquA from a protist to enable future structural studies.