Characterizing the Structure and Function of Rhodoquinone Biosynthesis Enzymes

Abstract

Rhodoquinone (RQ) is essential to the bioenergetics of many organisms that survive in low oxygen environments because it allows the electron transport chain to function with fumarate as a final electron acceptor instead of oxygen. Rhodoquinone biosynthesis protein a (RquA) is a putative methyltransferase-like enzyme that is essential for RQ biosynthesis in many bacteria and protists, making RquA a drug target specific to pathogens that require RQ. RquA uses ubiquinone as a substrate; however, the enzymatic mechanism of RquA is unknown. In this study we used Escherichia coli to produce RquA from Rhodospirillum rubrum, Blastocystis spp., Pygsuia biforma, and Euglena gracilis and purified it using Ni-NTA affinity chromatography under denaturing conditions. The recombinant RquA was initially refolded by shock dilution in Brij-35 (0.1% w/v) and we are further optimizing this refolding condition prior to protein crystallization. In addition, a computational approach was used to identify the possible cofactors and substrates required for RQ biosynthesis. The structure of RquA proteins were predicted using trRosetta to have a Rossmann-fold similar to that of S-adenosyl-L-methionine (SAM) dependent methyltransferases. Molecular dynamics simulation and the I-TASSER server predicted that SAM binds with RquA from Rhodospirillum via two aspartic acid residues. Mutation of those residues to alanine disrupted RQ production, confirming that SAM is required for RquA activity. Future computational studies will investigate possible mechanisms by which RquA localizes to cell membranes as well as devise strategies to increase the solubility of recombinantly produced RquA, in order to aid the in vitro characterization of RquA. Keywords: RquA; rhodoquinone; protein refolding; MD simulation; structure prediction.